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4-1-2010

Farnesol Signaling in Candida albicans

Melanie L. Langford
University of Nebraska at Lincoln, langford.melanie@gmail.com

Langford, Melanie L., "Farnesol Signaling in Candida albicans" (2010). Dissertations and Theses in Biological Sciences. Paper 9. http://digitalcommons.unl.edu/bioscidiss/9

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Farnesol Signaling in Candida albicans

By

Melanie L. Langford

A Dissertation

Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy

Major: Biological Sciences

Under the Supervision of Professor Audrey L. Atkin

Lincoln, Nebraska

May, 2010

Farnesol Signaling in Candida albicans Melanie L. Langford, Ph.D. University of Nebraska, 2010

Advisor: Audrey L. Atkin Candida albicans is a polymorphic fungus that causes a range of disease in humans, from mucosal infections to systemic disease. Its ability to cause disease is linked to conversion between yeast and filamentous forms of growth, and the first quorum-sensing molecule discovered in an eukaryote, farnesol, blocks this transition. In C. albicans, farnesol also kills mating-competent opaque cells, inhibits biofilm formation, protects the cells from oxidative stress, and can be a virulence factor or protective agent in disseminated and mucosal mouse models of infection, respectively. While much emphasis has been placed on determining its effect on C. albicans morphology, the molecular response to farnesol is not completely understood. The overall theme for this dissertation was to better understand the C. albicans molecular response to farnesol under quorum sensing conditions. Due to the duplicitous nature of the farnesol response in C. albicans, i.e., its ability to kill cells or simply alter morphology, we clearly defined the environmental conditions in which farnesol acts as a quorum sensing molecule or as a toxic agent towards C. albicans. This clarification enabled a subsequent two-pronged approach to study the molecular response to farnesol during morphological regulation. A direct approach was used to investigate the role of a likely candidate, Tup1, a negative regulator of hyphal development, in farnesol signaling. Secondly, a screening approach was utilized to identify new farnesol resistant mutants that may participate in the farnesol

response. From the mutants identified, Czf1 (C. albicans zinc finger) was selected for further characterization and was shown to play a vital role in the morphological response to farnesol as well as farnesol tolerance. Overall, this study identified two new factors involved in farnesol signaling, and highlights the power of farnesol as a tool with which to unravel the complex signaling networks present in C. albicans.

iv ACKNOWLEDGEMENTS No one ever gets through their Ph.D. program on their own, and I am certainly no exception. I would like to begin by thanking my wonderful advisor, Audrey Atkin. What a happy accident joining your lab turned out to be- I never would have guessed that I would switch from studying bacteriology to fungal biology! It makes an incredible difference to work for someone that not only encourages quality research but is so pleasant on a personal level as well, things that I will miss. During my time spent in your lab, I have learned nearly everything I know about fungi, and I have had the chance to learn several new lab techniques as well, such as working with RNA. I am happy you encouraged me to give several oral and poster presentations, both locally and nationally; what initially seems like more work always pays off in learning experiences. I also think it says a lot about the career development information you provide your lab members when I have heard nearly everything discussed in our Professionalism course this semester from you already over the course of my graduate program. Lastly I always appreciated your support whenever a crisis arose (be it professional, experimental, or personal). I would also like to extend my thanks to Kenneth Nickerson, my favorite fungal biology neighbor. I will miss having a chemistry expert right next door and your

enthusiasm for nearly everything and anything in science. I have truly enjoyed having joint Atkin-Nickerson lab meetings, and I feel they have been critical to the completion of my degree. To both Audrey and Ken, I am always amazed at how well your

collaboration works (given your entirely different research styles, personalities, etc), but I am happy that it does (maybe I fall somewhere in the middle of you two?). I think I have

v learned about balancing careful experimental design with exploratory experiments moving in new directions, and I have also enjoyed writing papers with both of you and learning about the process of paper submission. Next I would like to thank all my committee members: Steve Harris, Jack Morris, and Luwen Zhang. You always provided interesting ideas and suggestions during my committee meetings, and I appreciate that you always did so in a very constructive manner. Thanks to Cheryl Bailey for teaching me real-time PCR from scratch- it is easy to find people that will let you borrow their machines, but not so easy to find someone to teach you the method and theory behind it. There are also so many past and current lab members to thank. First, I would like to thank Bessie Kebaara, a former post-doc in the lab that I still miss having around. I could always go to you with technical questions, scientific ideas, or just to complain about the cold weather. Dhammika Navarathna and Raluca Dumitru, former Ph.D. students in the Nickerson lab, both taught me important Candida techniques and were great role models for me as a new student. And thanks to Suman Ghosh, another former Ph.D. student, for teaching me farnesol extraction techniques and for being a good friend. Of the current students in our research group, I would first like to extend my gratitude to Sahar Hasim, for being such an excellent collaborator on the farnesol toxicity project. You are such an amiable and reliable person to work with, and even though things took longer to finish than we originally thought (seems they always do), I think we learned some very valuable information over the course of that project. Thanks also to Swetha Tati (my technical support aide prior to my defense), Krista Fager, Jeff Bunker, and Narmin Tahirova for all your helpful comments and suggestions during journal club and lab meetings.

vi To my family: Mom, Dad, Courtney, Gina, Jim, and all the Langford brothers, I would like to thank you for all of your support during both my M.S. and Ph.D. degrees. Believe it or not, attempting to explain to you what I do in the lab has actually improved my oral presentations to more general scientific audiences! I am very lucky to have such supportive family members on my side. And lastly, I would like to thank my husband, Gabe. I am certain that I would not have been able to get through graduate school without you, much less have the confidence to even pursue my Ph.D. degree. It has been chaotic but fun to go through the ups and downs of graduate school with you, as you were always the perfect confidant. I am so excited to start the next phase of our life together with our new family.

vii TABLE OF CONTENTS ACKNOWLEDGEMENTS. ........................................................................................... iv TABLE OF CONTENTS ............................................................................................... vii LIST OF FIGURES ...........................................................................................................x LIST OF TABLES ........................................................................................................... xi

CHAPTER 1: Introduction..............................................................................................1 References..........................................................................................................................23 Figure Legends...................................................................................................................18 Figures................................................................................................................................19 Tables.................................................................................................................................20

CHAPTER 2: Activity and toxicity of farnesol on Candida albicans is dependent on growth conditions.............................................................................................................35 Abstract ..............................................................................................................................36 Introduction........................................................................................................................37 Methods..............................................................................................................................39 Results................................................................................................................................40 Discussion ..........................................................................................................................42 References..........................................................................................................................44 Figure Legends...................................................................................................................49 Figures................................................................................................................................50

viii CHAPTER 3: Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous growth induction .........................................................................................53 Abstract ..............................................................................................................................54 Introduction........................................................................................................................55 Methods..............................................................................................................................59 Results................................................................................................................................62 Discussion ..........................................................................................................................68 References..........................................................................................................................73 Figure Legends...................................................................................................................80 Figures................................................................................................................................82 Tables.................................................................................................................................87

CHAPTER 4: A novel role for Czf1 in farnesol tolerance and the morphological response to farnesol in Candida albicans .......................................................................89 Abstract ..............................................................................................................................90 Introduction........................................................................................................................91 Methods..............................................................................................................................94 Results................................................................................................................................97 Discussion ........................................................................................................................106 References........................................................................................................................111 Figure Legends.................................................................................................................119 Figures..............................................................................................................................122 Tables...............................................................................................................................130

ix CHAPTER 5: Summary and Future Directions ........................................................147 References........................................................................................................................157

x LIST OF FIGURES Figure 1-1...........................................................................................................................19 Figure 2-1...........................................................................................................................50 Figure 2-2...........................................................................................................................51 Figure 2-3...........................................................................................................................52 Figure 3-1...........................................................................................................................82 Figure 3-2...........................................................................................................................83 Figure 3-3...........................................................................................................................84 Figure 3-4...........................................................................................................................85 Figure 3-5...........................................................................................................................86 Figure 4-1.........................................................................................................................122 Figure 4-2.........................................................................................................................123 Figure 4-3.........................................................................................................................124 Figure 4-4................................................................................................................. 125-126 Figure 4-5.........................................................................................................................127 Figure 4-6.........................................................................................................................128 Figure 4-7.........................................................................................................................129

xi LIST OF TABLES Table 1-1. ..................................................................................................................... 20-21 Table 1-2 ............................................................................................................................22 Table 3-1 ............................................................................................................................87 Table 3-2 ............................................................................................................................88 Table 4-1 ..........................................................................................................................130 Table 4-2 ..........................................................................................................................131 Supplementary Table 4-1......................................................................................... 132-146

CHAPTER 1 INTRODUCTION

Reference: Langford, M.L., Atkin, A.L., and Nickerson, K.W. (2009) Cellular Interactions of Farnesol, a Quorum Sensing Molecule Produced by Candida albicans. Future Microbiol. 4: 1353-62.

2 INTRODUCTION The discovery of E,E-farnesol as a quorum sensing molecule in Candida albicans (Hornby et al., 2001) demonstrated the existence of quorum sensing in a eukaryotic organism, a process once thought to be confined to bacteria. The earlier work on the discovery and characterization of farnesol was reviewed by Nickerson et al (Nickerson et al., 2006). C. albicans is a polymorphic fungus that is capable of growing in yeast, hyphal, pseudohyphal, opaque, and chlamydospore cell morphologies. Farnesol is

capable of blocking the yeast to hyphal/pseudohyphal (filaments) switch (Hornby et al., 2001), a conversion that is the focus of intense study in C. albicans because of its role in virulence (Lo et al., 1997; Saville et al., 2003). Another quorum sensing molecule (QSM) described in C. albicans is farnesoic acid (Oh et al., 2001). Farnesoic acid, like farnesol, acts to block the yeast to filament transition (Oh et al., 2001), but it is far less active than farnesol (Shchepin et al., 2003) and is produced by only one known strain of C. albicans, 10231 (Hornby and Nickerson, 2004; Oh et al., 2001). Additional possible quorum sensing molecules are reviewed by Kruppa (Kruppa, 2009). Farnesol also blocks biofilm formation by C. albicans (Alem et al., 2006; Cao et al., 2005; Martins et al., 2007; Ramage et al., 2002) in a manner consistent with its inhibition of the yeast to filament conversion. The mating-competent form of C. albicans, opaque cells, can be adversely affected by farnesol; in aerobic conditions the cells die by necrosis (Dumitru et al., 2007) but remain unharmed in anaerobic conditions (Dumitru et al., 2004). Lastly, the production of chlamydospores, a C. albicans cell morphology with unknown function, is increased in the presence of very high (10 mM) levels of farnesol (Martin et al., 2005). Given that it may affect all five C. albicans growth morphologies, it has

3 become clear that farnesol plays an influential role in C. albicans physiology, and the search is underway to elucidate the molecular mechanism(s) of these effects. The Molecular Response to Farnesol in C. albicans The first described function of farnesol was to block filamentation induced by a variety of environmental signals (Hornby et al., 2001; Mosel et al., 2005), and several factors involved in the filamentation process are known to play a role in the C. albicans farnesol response. Ras1 of the cyclic AMP pathway, as discussed later, is a candidate for direct inhibition by farnesol (Davis-Hanna et al., 2008). Repressors of filamentation, Tup1 and Nrg1, also play critical roles in the farnesol response, as tup1/tup1 and nrg1/nrg1 mutants are unable to respond to farnesol (Kebaara et al., 2008). The presence of farnesol increases TUP1 expression and suppresses the haploinsufficient phenotype of a TUP1/tup1 mutant (Kebaara et al., 2008). A two-component signal transduction pathway histidine kinase, Chk1 (Kruppa et al., 2004), and mitogen activated protein (MAP) kinase pathway components, Cph1 and Hst1 (Sato et al., 2004), may also be involved in the response to farnesol, but Cph1 might play a downstream role since a cph1/cph1 mutant retains the ability to respond to farnesol (Davis-Hanna et al., 2008). Regulators of filamentation are not the only genes involved in the farnesol response. Microarray analyses have identified other categories of genes affected by farnesol treatment such as heat shock genes, drug resistance genes, cyclin and cell proliferation genes, histone genes, genes expressed at high cell density, phagocytosis response genes, and adhesion genes (Cao et al., 2005; Cho et al., 2007; Enjalbert and Whiteway, 2005; Uppuluri et al., 2007). With all of the different pathways and responses induced by farnesol, it promotes the question: Does farnesol have one or more specific target(s) in

4 the cell, or does it somehow elicit a more general and nonspecific response with pleomorphic consequences? Are there farnesol receptors or farnesol binding proteins? The morphological response to farnesol in C. albicans appears to be very sensitive to minor changes in the structure of farnesol, providing support for the specific target(s) model, rather than a scenario involving general membrane disruption. For example, there are four isomers of farnesol but only E,E-farnesol is capable of blocking filamentation in C. albicans (Shchepin et al., 2003). This conclusion is based on a comparison between pure (96%) E,E-farnesol and two commercial mixed isomers containing 56 and 36% E,E-farnesol. These three farnesol preparations reduced germ tube formation (early stage filaments) to 50% at 1.2, 3.5, and 4.4 M, respectively (Shchepin et al., 2003). Thus, there was sufficient E,E-farnesol in the two samples with mixed isomers to account for all of their QSM activities. Further, the 12-carbon backbone of farnesol appears to be critical for its ability to block filamentation because altering the chain length generally results in decreased filament inhibitory activity (Hogan et al., 2004; Kim et al., 2002; Shchepin et al., 2003; Shchepin et al., 2005). Other farnesol analogs and related molecules from other organisms, such as 3-oxo-C12-homoserine lactone and dodecanol, are capable of blocking filamentation in C. albicans, suggesting some limited flexibility for the inhibitory action of farnesol-related molecules (Davis-Hanna et al., 2008; Hogan et al., 2004; Kim et al., 2002; Shchepin et al., 2003; Shchepin et al., 2005). One, but not the sole explanation for these results is the presence of a farnesol receptor(s) or farnesol binding protein(s) in C. albicans. Fluorescent farnesol (Shchepin et al., 2005) and a

5 farnesol affinity column (Shchepin, 2006) were developed to help isolate farnesol binding proteins, so far with limited success. Farnesol and Ras1/cAMP Signaling Ras1 and the adenylyl cyclase-cAMP-protein kinase A (PKA)-Efg1 pathway (Fig. 1) have been proposed as a potential direct target for farnesol inhibition (Davis-Hanna et al., 2008). This idea is very reasonable and it is persuasive that farnesol was able to reverse filamentation in the dominant active (CAI4-RaslG13V) mutant of C. albicans and farnesol inhibition of filament formation was itself reversed by addition of dibutyrylcAMP (Davis-Hanna et al., 2008, discussed in more detail by Paula Sundstrom and Deborah Hogan in this issue). It may be important that Ras1 is one of the few

farnesylated proteins in C. albicans, and it has been estimated that the farnesylated, membrane-bound form of Ras1 is ca. 100 X more effective in activating adenylyl cyclase than is the cytoplasmic form (Kuroda et al., 1993). Thus, there are several ways by which farnesol could inhibit the Ras1-cAMP-PKA-Efg1 pathway (Fig. 1). A partial list includes: blocking the farnesylation of Ras1, see review by Hancock et al (Hancock, 2003), releasing farnesylated Ras1 from the membrane (Fig. 1A), binding directly to Ras1 (Fig. 1B), and binding directly to a downstream protein such as adenylyl cyclase (Fig. 1C). It is unlikely that farnesylated Ras1 has the farnesyl tail removed because it is attached by a very stable thioether bond and, where measured, farnesylated Ras1 has a long half life. Having Ras1 as a direct target for farnesol can provide some unity for many of the filamentation genes involved in the farnesol response since it is a common regulator for the Hst7/Cph1 MAP kinase pathway and the cAMP pathway (Castilla et al., 1998; Feng

6 et al., 1999; Leberer et al., 2001). While it is unknown what factors regulate Tup1/Nrg1 and Chk1, it is possible that they are also under the control of Ras1. A second possibility for farnesol signaling is the presence of multiple farnesol targets, each affecting different aspects of filamentation in response to different inducing stimuli, while a third possibility is that farnesol interacts with the cell membrane in a very specific manner to induce a pleiotropic signaling response in the rest of the cell. Notice that Figure 1 has been drawn with both intracellular and extracellular farnesol. This was done to emphasize that further research is warranted to determine the exact nature of the molecular response to farnesol during yeast to filament inhibition. It is difficult to say whether farnesol acts from the outside or the inside because it can cross the cytoplasmic membrane by diffusion. On the one occasion when cellular localization was attempted (Navarathna et al., 2005), the values for extracellular, intracellular, and membrane-associated farnesol were 0.115, 0.009, and 0.106 mg/g dry weight of cells, respectively (Navarathna et al., 2005). These values correspond to concentrations of ca. 4 M extracellular (Nickerson et al., 2006) and 13.5 M intracellular, with the intracellular calculation based on a yeast cytoplasmic volume of ca. 3 l/mg dry weight. Similarly, the membrane-associated farnesol would constitute 1-2% of the total extractable membrane lipids based on a value of 8.5 mg extractable lipid/g dry weight (Hitchcock et al., 1986). Possible Artifacts We have found several artifacts associated with the cellular response to farnesol. Caveats which must be considered include: 1) some auxotrophic mutants (his1/his1, arg4/arg4) produce germ tubes faster than their wild type, prototrophic counterparts and 2) as a consequence, when examining a mutant for its response to farnesol, it is

7 always safer to run a time course with and without farnesol until 90-100% germ tube formation has been achieved in the control lacking farnesol (our unpublished data). This prevents a misinterpretation for lack of farnesol response rather than a faster rate of filamentation. A third potential artifact is derived from farnesols lipophilic nature. We previously pointed out how ca. 150x more farnesol was needed to block germ tube formation in the presence of 10% serum than in its absence (Mosel et al., 2005). The farnesol concentration would be effectively reduced because of the lipid binding capacity of serum albumins. A similar trap could occur when farnesols action in blocking germ tube formation or inhibiting growth is reversed by added diacylglycerol (DAG) or 1oleoyl2-acetyl-sn-glycerol (OAG). Machida et al (Machida et al., 1999), Voziyan et al (Voziyan et al., 1995) and Uppuluri et al (Uppuluri et al., 2007) attributed internal modes of action, i.e. protein kinase C activation (Voziyan et al., 1995) or interference with phosphatidylinositol signaling (Machida et al., 1999; Uppuluri et al., 2007), to this DAG/OAG reversal. However, micelle forming detergents or lipids such as DAG and OAG could incorporate the farnesol and effectively reduce its concentration. Thus, a simple way of distinguishing between reversal by an external micelle trap versus a more specific cytoplasmic mechanism is to find out whether equivalent amounts of detergents such as Triton X-100 and NP40 also reverse farnesols action. This idea was recently tested for farnesols growth inhibition of Candida parapsilosis. OAG and Triton X-100 showed equivalent reversal of farnesols growth inhibition (T. Rossignol and G. Butler, personal communication), and in this case, no conclusions could be drawn on farnesols intracellular mode of action. Farnesol Production by Candida Species

8 Weber et al (Weber et al., 2008) recently compared the farnesol production levels from 56 strains of eight Candida species. C. albicans had the highest production levels (35 + 16 M) compared to the other Candida species tested: C. dubliniensis, C.

tropicalis, C. parapsilosis, C. guilliermondii, C. kefyr, C. krusei, and C. glabrata (Weber et al., 2008). The closely related species C. dubliniensis produced 8.7 + 3.8 M farnesol while the remaining Candida species all produced < 1 M farnesol. This value for C. albicans (35 M) is substantially higher than our best estimates (Nickerson et al., 2006) of 2 to 4 M farnesol at a yeast cell density of 108/ml, based on production of ~ 0.13 mg farnesol per g dry weight of fungus (Hornby and Nickerson, 2004). Production levels for two laboratory and four clinical isolates were tightly clustered from 0.11 to 0.14 mg per g dry weight (Hornby and Nickerson, 2004) whereas the seven strains of C. albicans studied by Weber et al (Weber et al., 2008) varied from 13 to 58 M farnesol. This wide variance (Weber et al., 2008) might be resolved by normalizing the data on a per g dry weight basis. Also, despite the different analytical procedures employed, GC/MS by Hornby and Nickerson (Hornby and Nickerson, 2004) and derivatization with 9-anthroyl nitrile followed by HPLC by Weber et al (Weber et al., 2008), the estimates of 35 M vs. 2-4 M farnesol may be compatible. If the cell densities achieved following 24 hrs growth in RPMI at 37C (Weber et al., 2008) were ca. 109/ml, compared to only 108/ml in GPP at 30C (Hornby and Nickerson, 2004), then the two data sets would be in remarkably close agreement. Careful consideration must also be given to the growth medium when measuring farnesol production levels. For example, the addition of 10% fetal calf serum (FCS) lowered farnesol production ca. 18-fold (Weber et al., 2008) but the mechanism behind

9 this reduction remains unclear. The nonspecific lipid binding capability of serum

albumin was evident in the fact that 150-fold more farnesol was needed to block filamentation than in its absence (Mosel et al., 2005). Similarly, industrial stratagems to maximize farnesol production (Muramatsu et al., 2008, 2009) commonly used an external lipid sink such as 5% soybean oil to shift the equilibrium toward production and excretion of farnesol. Thus, 10% FCS should have increased farnesol production rather than decreased it (Weber et al., 2008). Possibly the low levels of farnesol detected with 10% FCS used an extraction protocol which did not fully denature the lipid-binding albumins. The importance of a nearby lipid sink will be revisited in our discussion on the role of farnesol in pathogenicity. Manipulating the sterol biosynthetic pathway in C. albicans and other fungi with inhibitors such as fluconazole, terbinafine, SQAD, or zaragozic acid can result in increased farnesol production levels (Hornby et al., 2003; Hornby and Nickerson, 2004), and three genes have been identified in C. albicans thus far that regulate farnesol production: DPP3, TUP1, and NRG1 (Kebaara et al., 2008; Navarathna et al., 2007a). C. albicans Dpp3 is an ortholog of the S. cerevisiae phosphatase Dpp1 (Toke et al., 1998), and it presumably acts as a phosphatase to convert farnesyl pyrophosphate to farnesol (Navarathna et al., 2007a). Deletion of the C. albicans DPP3 gene results in farnesol production levels that are six times lower than wild type and parental levels (Navarathna et al., 2007a). Conversely, Tup1 and Nrg1 appear to play a negative role in farnesol production; tup1/tup1 and nrg1/nrg1 mutant strains produce 17 and 19fold higher farnesol, respectively, than their parents, but it is unclear whether this is a result of direct or indirect sterol biosynthesis regulation (Kebaara et al., 2008). Further

10 research is required to fully understand the regulation of farnesol production in both C. albicans and other Candida species. Farnesol and Interspecies Communication A developing branch of research has focused on the effects of farnesol signaling on interspecies communication. A brief summary of the effects of farnesol on other organisms is described in Table 1. Filament and biofilm development are each blocked by farnesol in the closely related C. dubliniensis, similar to the C. albicans morphological response to farnesol (Henriques et al., 2007; Jabra-Rizk et al., 2006b; Martins et al., 2007). In contrast, C. parapsilosis, which normally produces very low levels of farnesol, appears to respond in a different manner; farnesol reduces C. parapsilosis biofilm formation independently from filament inhibition (Laffey and Butler, 2005; Rossignol et al., 2007). The use of physiologically relevant farnesol concentrations to avoid

unintended cytotoxic effects will determine whether farnesol blocks dimorphism and/or biofilm formation in other Candida species. In other cell types, farnesol can be inhibitory or toxic, often accompanied by elevated reactive oxygen species (ROS) production. However, these reports encompass a huge range of farnesol concentrations, many of which are above the ca. 1 mM solubility limit for farnesol, increasing the likelihood that the responses observed are both specific and non-specific, or detergent-like. Given that the production of extracellular signaling molecules is certainly not unique to C. albicans, there has also been some recent work describing the converse situation in which the response of C. albicans to bacterial signaling molecules is examined (Table 2). It is important to distinguish between general growth inhibition (an antifungal antibiotic) and specific inhibition of one type of growth, e.g. a QSM blocking

11 the yeast to filament transition in C. albicans. For instance (Table 2), while Burkholderia diffusible signal factor (BDSF) at a 5 M concentration inhibited 70% of germ tube formation for C. albicans cells, that same concentration also strongly inhibited growth (Boon et al., 2008). It is intriguing how a similar molecule produced by Xanthomonas campestris, diffusible signal factor (DSF), can inhibit filamentation without any growth inhibition (Boon et al., 2008; Wang et al., 2004), even though the two molecules differ by only one methyl group. Molecules that are not inhibitory for the general growth of C. albicans, yet induce a signaling response are legitimate candidates for cross-kingdom signaling molecules (Table 2). An interesting consideration to make when evaluating the variety of responses to QSMs such as farnesol in different organisms is the likelihood of the cells to interact with one another during the commensal state versus during an infection. Similarly, one must also consider the location of the interaction within the host, as farnesol production by C. albicans results in different host responses depending on the site of infection. For instance, it is likely important for C. albicans commensal life style in the gastrointestinal tract (Kumamoto and Vinces, 2005b) that farnesol production is turned off during anaerobic growth (Dumitru et al., 2004). Lastly, farnesol must be used carefully and at the appropriate physiologically relevant concentrations in order to avoid artifactual observations, such as unintended cytotoxic effects. Farnesol-Mediated Cell Death in C. albicans With regard to the possibility of farnesol-mediated cell death, the literature has conflicting reports on the appropriate levels of farnesol with which to treat C. albicans. Several groups have used 150-250 M farnesol with no apparent cell death (Davis-Hanna

12 et al., 2008; Henriques et al., 2007; Hogan et al., 2004; Kruppa et al., 2004); one study by Jabra-Rizk et al (Jabra-Rizk et al., 2006b) concluded the minimum inhibitory concentration (MIC) for C. albicans was >250 M while another study concluded that 40 M farnesol induced cell death (Shirtliff et al., 2009). Conflicts have also emerged regarding the concentration of farnesol needed to block filamentation in C. albicans, with effective concentration reports ranging from 4 M (Mosel et al., 2005), up to 250 M (Kruppa et al., 2004). Another important finding was that of Davis-Hanna et al (DavisHanna et al., 2008) who noted that higher concentrations of farnesol were required to block filamentation in plastic microtiter plates compared to borosilicate glass tubes or flasks, suggesting a possible farnesol adsorption effect by plastic that might lower the effective concentration of farnesol. Our summary view is that the appropriate level of farnesol to use for filament inhibition is 50 M farnesol when using glassware; this corresponds to physiologically relevant concentrations of farnesol produced by stationary-phase C. albicans cultures (Weber et al., 2008). While the plastic versus glassware variable may explain some discrepancies in the literature, additional variables need to be considered when evaluating farnesol-mediated cell death in C. albicans. Farnesol is clearly a bioactive molecule. With the caveat that inappropriately high concentrations were often used, the recurrent theme in Table 1 is that farnesol is inhibitory or lethal to a great many other cell types. Thus, a fundamental question is how C. albicans has evolved to withstand the farnesol which it produces in great abundance. This question is exactly analogous to an antibiotic producing strain being resistant to that antibiotic. A corollary to this idea is that reports of farnesol mediated cell death be

13 viewed as circumstances where C. albicans has let down its guard, i.e. the molecular defense mechanisms are regulable rather than constant or intrinsic. Farnesol has been reported to cause growth defects (Uppuluri et al., 2007), necrosis (Dumitru et al., 2007) and apoptosis (Shirtliff et al., 2009) in C. albicans, although the precise concentrations required are somewhat unclear, because different conditions were used to grow the cells and different methods were used to assess cell damage. Work is underway in our laboratories to investigate farnesol tolerance by C. albicans, taking into account many environmental variables, in an effort to unify and validate existing data in the literature (Langford et al, data not shown). Our conclusions so far are that four factors are significant. First, only white cells of C. albicans are resistant. Dumitru et al (Dumitru et al., 2007) observed that the opaque cells were very sensitive to farnesol, being lysed rapidly by 40 M. Following this line of thinking, in a saturated aerobically grown culture of predominantly white cells, the farnesol levels could possibly reach levels high enough to kill opaque cells. Second, farnesol resistance is somewhat energy dependent. The farnesol-induced death and apoptosis reported by Shirtliff et al (Shirtliff et al., 2009) was for cells which had been stored for 24h in PBS buffer with no exogenous energy sources. Third, as was first pointed out by Uppuluri et al (Uppuluri et al., 2007), the starting growth phase of the inoculum is critical. At 25C, C. albicans log-phase cells were considerably more sensitive to growth inhibition by farnesol than were stationary-phase cells. Stationary-phase inocula maintained similar growth rates and viability with 0-100 M farnesol, while log-phase cells grew considerably slower with 40 M farnesol and viability was only 18% with 100 M farnesol added (Uppuluri et al., 2007). Fourth, ROS are generated during farnesol

14 treatment of both white and opaque cells, and evidence supports mitochondrial perturbation when cytotoxic levels of farnesol are present (Dumitru et al., 2007; Shirtliff et al., 2009; Uppuluri et al., 2007). The importance of ROS is also implicit in the findings of Dumitru et al (Dumitru et al., 2007); under anaerobic conditions C. albicans tolerated mM levels of farnesol. Careful use of physiologically relevant farnesol

concentrations combined with clear indications of the previously mentioned variables will hopefully prevent future confusion on this issue. Is farnesol-mediated death in C. albicans induced in a specific or non-specific manner? Evidence exists supporting both models. Ras1/cyclic AMP signaling again becomes relevant as it is known that an activated Ras-cAMP-PKA pathway and cAMPstimulatory drugs promoted apoptotic cell death in C. albicans (Phillips et al., 2006). However, the connection of farnesol to apoptosis (Shirtliff et al., 2009) is less clear. Based on the findings of Phillips et al (Phillips et al., 2006), one would expect farnesol to lessen apoptosis rather than to promote it. Additionally, the lysis of opaque cells by farnesol was not apoptotic (Dumitru et al., 2007). Using the same procedures as had been used for showing apoptosis in A. nidulans (Semighini et al., 2006), including the definitive Annexin V test (Semighini et al., 2006), we were unable to demonstrate apoptosis in farnesol lysed opaque cells (Dumitru and Nickerson, unpublished data). Since farnesol is lipophilic, it has the ability to disrupt cell membranes, and this supports the nonspecific-mediated killing model. Understanding how farnesol regulates C.

albicans growth morphologies, growth rates, and even cell death, may be essential components for understanding the role of farnesol in the host. The Role of Farnesol In Vivo

15 Our final topic is the role of farnesol during infection. At the time of its discovery as a QSM, two ideas were put forward regarding its in vivo importance (Hornby et al., 2001). One was that, due to its ability to block the yeast to filament transition, farnesol treatment would act as a therapeutic in a mouse model of infection. The other was that farnesol would act as a virulence factor. Research on the topic has shown that the role of farnesol in vivo is not so simplistic. Navarathna et al (Navarathna et al., 2005;

Navarathna et al., 2007a) showed that farnesol acts as a virulence factor in the mouse tail vein injection assay, leading to the question of just how much farnesol is produced by C. albicans in the mouse. A definitive answer to this question will be difficult. The in vitro production estimates were for cells grown in glass flasks whereas in vivo production would be for cells growing in close proximity to mouse membranes of many types which could easily act as external lipid sinks, thus enhancing farnesol production. C. albicans is capable of evading part of the host immune system by escaping from macrophages through the production of filaments (Ghosh et al., 2009; Lorenz et al., 2004). While external farnesol reduces the viability of macrophages in vitro (Abe et al., 2009), the interesting question remains whether farnesol levels naturally produced by C. albicans are high enough to affect macrophage function/viability and thus enable fungal survival and escape from the macrophage. Does farnesol production in the phagolysosome contribute to escape from the macrophage? Experiments which add exogenous farnesol to the macrophage cannot answer this question. Also, an unfortunate obstacle for answering this question directly with the dpp3/dpp3 mutant currently available is twofold. First, it is knocked out in only one of two genes which convert farnesyl pyrophosphate to farnesol and thus it still produces 15% as much farnesol as its

16 parent and, second, it is an arginine auxotroph (Navarathna et al., 2007a). Ghosh et al (Ghosh et al., 2009) demonstrated the requirement of arginine biosynthesis by C. albicans to effectively penetrate and escape murine macrophages. Briefly (Fig 1), Ghosh et al (Ghosh et al., 2009) showed that the rapid upregulation of arginine biosynthetic genes following macrophage ingestion, originally noted by Lorenz et al (Lorenz et al., 2004), occurred so that the arginine could be degraded to urea and then to CO2, a known signal for hyphal switching (Bahn and Muhlschlegel, 2006) necessary for C. albicans to escape. Therefore, the dpp3/dpp3 mutant and its parental strain SN152 are unable to escape from macrophages, regardless of the presence of DPP3. New prototrophic

mutants defective in farnesol production must be constructed before this question can be answered. Consistent with early hypotheses, Hisajima et al (Hisajima et al., 2008) recently published the finding that farnesol has a protective role against C. albicans in an oral model of murine infection. This presents an intriguing conundrum: how does farnesol enhance infection for disseminated candidiasis (Navarathna et al., 2007a) while treating infection for mucosal (oral) candidiasis? A similar conundrum is how farnesol can act externally to protect yeast cells from oxidative stress (Westwater et al., 2005) but also inhibit growth by enhancing ROS production by mitochondria (Machida et al., 1998b). This suggests other factors are at play other than or in addition to the effect of farnesol on C. albicans, potentially the hosts response to farnesol. Acknowledgements We wish to thank Geraldine Butler, Tristan Rossignol, Suman Ghosh, and Raluca Dumitru for allowing us to cite their unpublished data, as well as Deborah Hogan for

17 helpful reading of the manuscript. This work was supported by a faculty seed grant from the Constance Miriam and Ethel Corrine Syford Memorial Fund (to A.L.A.), the Hammond-Maude Fling Fellowship (to M.L.L.), and the Farnesol and Candida albicans Research Fund, University of Nebraska Foundation (to K.W.N.). These authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

18 Figure legends Figure 1-1. Farnesol impacts the cyclic AMP pathway in C. albicans. Three possible points of inhibition by farnesol.

19 Figure 1-1 Farnesol

A.

Ras1 NH3 HCO3Cyr1

B.

Farnesol

C.

Dur1,2 cAMP

L-ornithine

Urea Tpk1/2

Car1 Efg1 L-arginine

Hyphal-Specific Genes

20 Table 1-1. The effects of farnesol treatment in other cell types.

Organism C. dubliniensis

C. parapsilosis

Acinetobacter baumannii Aspergillus fumigatus Aspergillus nidulans

Farnesol Concentration(s) Summary of Farnesol Response Used 1.5 nM- 300 M Filament inhibition, no effect on growth, (Henriques et al., 2007; Martins et al., 2007), biofilm inhibition, MIC = 200M, lowered tolerance of fluconazole (Jabra-Rizk et al., 2006b) 1.5 nM-100 M Biofilm inhibition (Laffey and Butler, 2005), growth arrest 50 M, lipid metabolism, ribosome biogenesis, and amino acid biosynthesis genes affected, altered lipid polarization (Rossignol et al., 2007) 50 M- 200 M Growth inhibition (Peleg et al., 2008) 10 M - 250 M 1 M - 250 M Growth inhibition, apoptosis (Semighini et al., 2006) Apoptosis and ROS production (10 250 M) (Semighini et al., 2006), hyphal inhibition (1-10 M) (Semighini et al., 2006), autophagy, lipid metabolism, amino acid response, transcription, and translation genes affected (10-100 M) (Savoldi et al., 2008) Inhibition of conidiation (Lorek et al., 2008)

Aspergillus niger Fusarium graminearum Human gingival cells Human oral carcinoma cells Murine macrophages Paracoccidiodes brasiliensis Pseudomonas aeruginosa Saccharomyces cerevisiae

100 M- 10mM

100 M - 300 Apoptosis, altered spore germination and lysis M (Semighini et al., 2008) 10 M- 300 M Reduced proliferation and adhesion (Saidi et al., 2006) 10 M - 60 M Apoptosis (Scheper et al., 2008) 56 M - 112 M 5 M - 300 M 30 M - 300 M Apoptosis, ROS production, decreased phagocytic activity (Abe et al., 2009) Inhibition of yeast to hyphal and hyphal to yeast growth at low concentrations, growth inhibition at high concentrations (Derengowski et al., 2009) Inhibition of Pseudomonas quinolone signal (PQS) and pyocyanin production, decreased swarming motility, (Cugini et al., 2007; McAlester et al., 2008) Growth inhibition by cell cycle arrest, effect on mitochondria and increased reactive oxygen species (ROS) production (Machida et al., 1998a; Machida et al., 1999), cell death (Fairn et al.,

5 M- 5 mM

21 2007) Cell death, membrane disruption, biofilm inhibition, lipase inhibition, increased sensitivity to antibiotics (Jabra-Rizk et al., 2006a; Kuroda et al., 2007; Togashi et al., 2008) Cell death (Gomes et al., 2009) Growth inhibition, biofilm inhibition, prevention of dental caries in rats when in combination with fluoride and apigenin (Koo et al., 2002a; Koo et al., 2002b; Koo et al., 2003; Koo et al., 2005) Cell death (Hemmerlin and Bach, 2000; Hemmerlin et al., 2006)

Staphylococcus aureus Staphylococcus epidermidis Streptococcus mutans Tobacco (Nicotiana tabacum L. cv Bright Yellow2)

30 M - 4.5 mM

100 M -200 M 250 M - 5 mM

5 M -150 M

22 Table 1-2. The effects of secreted bacterial molecules on C. albicans.

Bacterium Acinetobacter baumannii Burkholderia cenocepacia P. aeruginosa

P. aeruginosa Salmonella enterica serovar Typhimurium Streptococcus gordonii Xanthomonas campestris

Secreted Molecule(s) Unknown, found in cell-free supernatant Cis-2-dodecenoic acid (Burkholderia diffusible signal factor, BDSF) 3-oxo-C12homoserine lactone, dodecanol, and C12acyl homoserine lactone Phenazines Unknown, found in cell-free supernatant Unknown, found in cell-free supernatant and missing/reduced in luxS mutant Cis-11-methyl-2dodecenoic acid (diffusible signal factor, DSF)

Summary of C. albicans Response Filament inhibition, biofilm inhibition (Peleg et al., 2008) Growth inhibition and filament inhibition (Boon et al., 2008) Filament inhibition and reversion to yeast morphology (Davis-Hanna et al., 2008; Hogan et al., 2004) Cell death (Gibson et al., 2009) Filament inhibition, biofilm inhibition, and reduced viability (Tampakakis et al., 2009) Induction of filamentous growth, suppression of farnesol-mediated filament inhibition (Bamford et al., 2009) Filament inhibition (Boon et al., 2008; Wang et al., 2004)

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33 Shirtliff, M.E., Krom, B.P., Meijering, R.A., Peters, B.M., Zhu, J., Scheper, M.A., Harris, M.L., and Jabra-Rizk, M.A. (2009) Farnesol-induced apoptosis in Candida albicans. Antimicrob Agents Chemother 53: 2392-2401. Tampakakis, E., Peleg, A.Y., and Mylonakis, E. (2009) Interaction of Candida albicans with an intestinal pathogen, Salmonella enterica serovar Typhimurium. Eukaryot Cell 8: 732-737. Togashi, N., Inoue, Y., Hamashima, H., and Takano, A. (2008) Effects of two terpene alcohols on the antibacterial activity and the mode of action of farnesol against Staphylococcus aureus. Molecules 13: 3069-3076. Toke, D.A., Bennett, W.L., Dillon, D.A., Wu, W.I., Chen, X., Ostrander, D.B., Oshiro, J., Cremesti, A., Voelker, D.R., Fischl, A.S., and Carman, G.M. (1998) Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase. J Biol Chem 273: 3278-3284. Uppuluri, P., Mekala, S., and Chaffin, W.L. (2007) Farnesol-mediated inhibition of Candida albicans yeast growth and rescue by a diacylglycerol analogue. Yeast 24: 681-693. Voziyan, P.A., Haug, J.S., and Melnykovych, G. (1995) Mechanism of farnesol cytotoxicity: further evidence for the role of PKC-dependent signal transduction in farnesol-induced apoptotic cell death. Biochem Biophys Res Commun 212: 479486. Wang, L.H., He, Y., Gao, Y., Wu, J.E., Dong, Y.H., He, C., Wang, S.X., Weng, L.X., Xu, J.L., Tay, L., Fang, R.X., and Zhang, L.H. (2004) A bacterial cell-cell

34 communication signal with cross-kingdom structural analogues. Mol Microbiol 51: 903-912. Weber, K., Sohr, R., Schulz, B., Fleischhacker, M., and Ruhnke, M. (2008) Secretion of E,E-farnesol and biofilm formation in eight different Candida species. Antimicrob Agents Chemother 52: 1859-1861. Westwater, C., Balish, E., and Schofield, D.A. (2005) Candida albicans-conditioned medium protects yeast cells from oxidative stress: a possible link between quorum sensing and oxidative stress resistance. Eukaryot. Cell 4: 1654-1661.

35

CHAPTER 2 Activity and toxicity of farnesol on Candida albicans is dependent on growth conditions

Reference: Langford, M.L., Hasim, S., Nickerson, K.W., and Atkin, A.L. (2010) Activity and toxicity of farnesol on Candida albicans is dependent on growth conditions. Antimicrob. Agents Chemother. 54(2): 940-2.

36 Abstract Farnesol interacts with Candida albicans as both a quorum sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size,

temperature, and media were altered. Parameters for farnesol tolerance/sensitivity are defined, validating previous studies and identifying new variables, such as energy sources. This study provides a clear understanding of what farnesol concentrations are lethal to C. albicans, based on environmental conditions.

37 Introduction Candida albicans is a dimorphic fungus of great medical importance. It is also the model system for studying quorum sensing for fungal dimorphism (Hornby et al., 2001). C. albicans excretes a quorum sensing molecule (QSM) called farnesol and when extracellular levels exceed a threshold of 1-5 M (Mosel et al., 2005), farnesol blocks the yeast-to-filament conversion. One feature of farnesols action as a QSM was that

exogenous farnesol up to 200-300 M does not alter the growth rate; instead, the cells grow as yeasts rather than as filaments. These results were quickly confirmed by

Ramage et al (Ramage et al., 2002), Hogan et al (Hogan et al., 2004), and Kruppa et al (Kruppa et al., 2004). Farnesol also impacts other aspects of C. albicans biology: it blocks biofilm formation (Ramage et al., 2002), acts as a virulence factor during systemic infection (Navarathna et al., 2007a), and is a protective factor during mucosal infection (Hisajima et al., 2008). Farnesol production is regulated in that it is turned off in opaque cells (Dumitru et al., 2007) and during anaerobic growth (Dumitru et al., 2004) but elevated in some mutants which are altered in morphology (Jensen et al., 2006) or locked in the filamentous morphology (Kebaara et al., 2008). Farnesol production also

increases in the presence of sublethal levels of sterol biosynthesis inhibitors (Hornby and Nickerson, 2004; Navarathna et al., 2005). Farnesol is a bioactive molecule with mild detergent-like properties. At

concentrations in the 20-50 M range it has been reported to inhibit or induce cell death in Saccharomyces cerevisiae (Machida et al., 1998a; Machida et al., 1999), Aspergillus nidulans (Semighini et al., 2006), Fusarium graminearum (Semighini et al., 2008), Paracoccidioides brasiliensis (Derengowski et al., 2009), and C. albicans opaque cells

38 (Dumitru et al., 2007). Similarly, farnesol has been reported to trigger cell death in several mammalian cell lines (Abe et al., 2009; Saidi et al., 2006; Scheper et al., 2008) and bacteria (Gomes et al., 2009; Jabra-Rizk et al., 2006a; Koo et al., 2003; Togashi et al., 2008), and it induces cytokine production, such as IL-6, by macrophages. Thus, the view to this point was that C. albicans exhibited exceptional tolerance to farnesol, as a necessary corollary to its production as an antagonistic molecule. This view was recently challenged by Shirtliff et al (Shirtliff et al., 2009) who reported that farnesol, at concentrations as low as 40 M, killed C. albicans by inducing apoptosis. Thus, in the spirit of constructive dialogue, we draw attention to the differences between the growth conditions used in our previous body of work under which farnesol would act as a signaling molecule, innocuous to growth, and the conditions under which Shirtliff et al (Shirtliff et al., 2009) observed cell death. Previous studies examining C. albicans sensitivity to farnesol (Dumitru et al., 2007; Jabra-Rizk et al., 2006b; Shirtliff et al., 2009; Uppuluri et al., 2007) have used vastly different assay conditions (varying temperatures, media, inoculum sizes, and inoculum growth phases), adding to confusion on the matter of farnesol-induced cell death. Critically, Shirtliff et al (Shirtliff et al., 2009) used cells that were grown

overnight, washed and resuspended in PBS for farnesol sensitivity assays. Since it is well known that detergent resistance in bacteria is an energy dependent process (Aspedon and Nickerson, 1993; Rajagopal et al., 2003), we were particularly interested whether farnesol resistance in C. albicans is similarly energy dependent.

39 Methods We examined farnesol sensitivity under a series of different growth conditions in plastic microtiter dishes. Variables included temperature, cell density, growth medium (rich vs. minimal), and inoculum history. We followed cell growth by means of optical density and cell death by methylene blue staining (Gibson et al., 2009). C. albicans cells were grown to stationary phase (unbudding cells; cultures were inoculated to an OD600 =0.1 and grown at 30C for 16-18h) or mid-log phase (OD600 =0.5), washed three times in PBS, and inoculated at the indicated levels with variable concentrations of farnesol. We used 10 mM and 100 mM stocks of E, E-farnesol in methanol so that the final methanol concentration never exceeded 1%, and this level of methanol had no effect on cell growth or cell death (data not shown).

40 Results In rich growth medium (YPD), no cell death was observed at farnesol concentrations up to 300 M and growth inhibition was only observed with 300 M farnesol (data not shown). When we switched to a defined glucose-proline (GPP pH6.8) (Kebaara et al., 2008)medium (Fig. 1), very similar growth curves were observed with all concentrations of farnesol up to 300 M when starting with stationary phase inocula (Fig. 1A). The cell growth experiments (Fig. 1) were simultaneously examined for cell death by staining with methylene blue (Fig. 2B, 2D). Minimal cell death occurred in GPP with a stationary phase inoculum, our standard growth conditions (Hornby et al., 2001; Mosel et al., 2005), and up to 300 M farnesol (Fig. 2D) consistent with growth curves seen in Fig. 1A. However, when we used inocula of exponentially growing cells, 40 M farnesol partially inhibited growth and further inhibition correlated with increasing farnesol levels (Fig. 1B). Log phase cells were killed by 100 and 300 M farnesol in GPP (Fig. 2B), consistent with the delayed growth seen in Fig. 1B. These results support the growth phase-dependent sensitivity described by Uppuluri et al (Uppuluri et al., 2007). Temperature does not play a prominent role in farnesol growth inhibition because we obtained similar growth curves at 25C, 30C, and 37C (data not shown). To examine the effects of different media on farnesol sensitivity, cells were compared under both growth (GPP) and storage (PBS) conditions, using both exponential and stationary phase inocula (Fig. 2). For exponential phase cells inoculated in PBS, even low levels of farnesol, i.e. 40 M, caused cell death (Fig. 2A), consistent with the findings of Shirtliff et al (Shirtliff et al., 2009). The cells in PBS were far more sensitive to farnesol when they had come from an exponential phase inoculum than a stationary

41 phase inoculum (Fig. 2A, 2C). Interestingly, both exponential and stationary phase cells showed increased tolerance to farnesol when incubated in growth media (GPP or YPD), compared to PBS (Fig. 2). As with growth rates, similar results for cell death were obtained at all 3 temperatures tested, 25C, 30C, and 37C. These observations suggest a role for energy source(s) in C. albicans farnesol tolerance. The previous experiments (Figs. 1 and 2) were conducted in 96 well plates with farnesol added at time zero to washed cells. Because of a possible farnesol adsorption effect with plastic (Davis-Hanna et al., 2008), we confirmed the farnesol sensitivity of exponentially growing cells in glass flasks (Fig. 3). We compared the farnesol sensitivity of C. albicans cells in exponential phase (OD600 = 0.5, Fig. 3A, 3B) and stationary phase (OD600 = 4.0, Fig. 3C, 3D) by adding farnesol directly to unwashed growing cultures. The results with glass flasks (Fig. 3) were consistent with those obtained with plastic 96 well plates.

42 Discussion A distinct benefit of this controversy is to focus attention on the conditions in which C. albicans tolerates farnesol while so many other cell types are killed by it. Stationary phase C. albicans cells exhibit extreme farnesol tolerance, even at 100-300 M farnesol, and these levels are lethal to most other organisms tested. Throughout, cell death was not accompanied by cell lysis because there was no drop in OD600 and the methylene blue positive cells remained intact. Similarly, optical densities of cells in PBS were not significantly affected by the presence of farnesol (data not shown). This lack of cell lysis with white cells of C. albicans is in marked contrast with opaque cells where 40 M farnesol caused rapid cell lysis (Dumitru et al., 2007). The differences in

susceptibility between white and opaque cells (Dumitru et al., 2007), aerobic and anaerobic cells (Dumitru et al., 2004), stationary and exponential cells [(Uppuluri et al., 2007) and Figs. 1-3], and between cells in buffer alone versus minimal media suggest a physiological adaptation to farnesol, depending on environmental conditions. Two places where this selective farnesol tolerance might be localized are the mitochondria, where farnesol stimulates ROS production in C. albicans and S. cerevisiae (Machida et al., 1998a; Machida et al., 1999; Shirtliff et al., 2009), and the lipid portion of the cytoplasmic membrane. Changes in fatty acid composition, sterol-rich domains (Alvarez et al., 2007), sphingolipids (Brown and London, 2000), and GPI-anchored proteins preferentially associated with lipid rafts (Brown and London, 2000) could be involved, as supported by the lipid domain alteration seen in C. parapsilosis after farnesol treatment (Rossignol et al., 2007). Although the mechanisms(s) involved in farnesol tolerance

43 remain unclear, conditions in which C. albicans is either sensitive or resistant to farnesol have now been outlined. In summary, stationary phase cells inoculated into minimal or rich media are ideal candidates for studying farnesol signaling, as these do not undergo cell death with high farnesol concentrations. This generalization was true for all three commonly used

temperatures (25C, 30C, 37C) as well as for experiments with starting cell densities of OD600 = 0.05 or 0.10. Conversely, optimal conditions to examine farnesol-mediated cell death include the use of log phase cells in PBS, under energy-starved conditions. Understanding these environmental parameters may unify many previous discrepancies in the literature and provide unambiguous conditions to induce each of farnesols unique effects on C. albicans.

Acknowledgements This work was supported by a faculty seed grant from the Constance Miriam and Ethel Corrine Syford Memorial Fund (to A.L.A.), the Hammond-Maude Fling Fellowship (to M.L.L.), and the Farnesol and Candida albicans Research Fund, University of Nebraska Foundation (to K.W.N.).

44 References Abe, S., Tsunashima, R., Iijima, R., Yamada, T., Maruyama, N., Hisajima, T., Abe, Y., Oshima, H., and Yamazaki, M. (2009) Suppression of anti-Candida activity of macrophages by a quorum-sensing molecule, farnesol, through induction of oxidative stress. Microbiol Immunol 53: 323-330. Alvarez, F.J., Douglas, L.M., and Konopka, J.B. (2007) Sterol-rich plasma membrane domains in fungi. Eukaryot Cell 6: 755-763. Aspedon, A., and Nickerson, K.W. (1993) A two-part energy burden imposed by growth of Enterobacter cloacae and Escherichia coli in sodium dodecyl sulfate. Can J Microbiol 39: 555-561. Brown, D.A., and London, E. (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275: 17221-17224. Davis-Hanna, A., Piispanen, A.E., Stateva, L.I., and Hogan, D.A. (2008) Farnesol and dodecanol effects on the Candida albicans Ras1-cAMP signalling pathway and the regulation of morphogenesis. Mol Microbiol 67: 47-62. Derengowski, L.S., De-Souza-Silva, C., Braz, S.V., Mello-De-Sousa, T.M., Bao, S.N., Kyaw, C.M., and Silva-Pereira, I. (2009) Antimicrobial effect of farnesol, a Candida albicans quorum sensing molecule, on Paracoccidioides brasiliensis growth and morphogenesis. Ann Clin Microbiol Antimicrob 8: 13. Dumitru, R., Hornby, J.M., and Nickerson, K.W. (2004) Defined anaerobic growth medium for studying Candida albicans basic biology and resistance to eight antifungal drugs. Antimicrob. Agents Chemother. 48: 2350-2354.

45 Dumitru, R., Navarathna, D.H., Semighini, C.P., Elowsky, C.G., Dumitru, R.V., Dignard, D., Whiteway, M., Atkin, A.L., and Nickerson, K.W. (2007) In vivo and in vitro anaerobic mating in Candida albicans. Eukaryot. Cell 6: 465-472. Gibson, J., Sood, A., and Hogan, D.A. (2009) Pseudomonas aeruginosa-Candida albicans interactions: localization and fungal toxicity of a phenazine derivative. Appl Environ Microbiol 75: 504-513. Gomes, F.I., Teixeira, P., Azeredo, J., and Oliveira, R. (2009) Effect of farnesol on planktonic and biofilm cells of Staphylococcus epidermidis. Curr Microbiol 59: 118-122. Hisajima, T., Maruyama, N., Tanabe, Y., Ishibashi, H., Yamada, T., Makimura, K., Nishiyama, Y., Funakoshi, K., Oshima, H., and Abe, S. (2008) Protective effects of farnesol against oral candidiasis in mice. Microbiol Immunol 52: 327-333. Hogan, D.A., Vik, A., and Kolter, R. (2004) A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology. Mol. Microbiol. 54: 12121223. Hornby, J.M., Jensen, E.C., Lisec, A.D., Tasto, J.J., Jahnke, B., Shoemaker, R., Dussault, P., and Nickerson, K.W. (2001) Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl. Environ. Microbiol. 67: 29822992. Hornby, J.M., and Nickerson, K.W. (2004) Enhanced production of farnesol by Candida albicans treated with four azoles. Antimicrob Agents Chemother 48: 2305-2307.

46 Jabra-Rizk, M.A., Meiller, T.F., James, C.E., and Shirtliff, M.E. (2006a) Effect of farnesol on Staphylococcus aureus biofilm formation and antimicrobial susceptibility. Antimicrob Agents Chemother 50: 1463-1469. Jabra-Rizk, M.A., Shirtliff, M., James, C., and Meiller, T. (2006b) Effect of farnesol on Candida dubliniensis biofilm formation and fluconazole resistance. FEMS Yeast Res 6: 1063-1073. Jensen, E.C., Hornby, J.M., Pagliaccetti, N.E., Wolter, C.M., Nickerson, K.W., and Atkin, A.L. (2006) Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens. Genome 49: 346-353. Kebaara, B.W., Langford, M.L., Navarathna, D.H., Dumitru, R., Nickerson, K.W., and Atkin, A.L. (2008) Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction. Eukaryot Cell 7: 980-987. Koo, H., Hayacibara, M.F., Schobel, B.D., Cury, J.A., Rosalen, P.L., Park, Y.K., VaccaSmith, A.M., and Bowen, W.H. (2003) Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol. J Antimicrob Chemother 52: 782-789. Kruppa, M., Krom, B.P., Chauhan, N., Bambach, A.V., Cihlar, R.L., and Calderone, R.A. (2004) The two-component signal transduction protein Chk1p regulates quorum sensing in Candida albicans. Eukaryot. Cell 3: 1062-1065. Machida, K., Tanaka, T., Fujita, K., and Taniguchi, M. (1998) Farnesol-induced generation of reactive oxygen species via indirect inhibition of the mitochondrial electron transport chain in the yeast Saccharomyces cerevisiae. 180 17.

47 Machida, K., Tanaka, T., Yano, Y., Otani, S., and Taniguchi, M. (1999) Farnesol-induced growth inhibition in Saccharomyces cerevisiae by a cell cycle mechanism. Microbiology 145: 293-299. Mosel, D.D., Dumitru, R., Hornby, J.M., Atkin, A.L., and Nickerson, K.W. (2005) Farnesol concentrations required to block germ tube formation in Candida albicans in the presence and absence of serum. Appl. Environ. Biol. 71: 49384940. Navarathna, D.H., Hornby, J.M., Hoerrmann, N., Parkhurst, A.M., Duhamel, G.E., and Nickerson, K.W. (2005) Enhanced pathogenicity of Candida albicans pre-treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis. J. Antimicrob. Chemother. 56: 1156-1159. Navarathna, D.H., Hornby, J.M., Krishnan, N., Parkhurst, A., Duhamel, G.E., and Nickerson, K.W. (2007) Effect of farnesol on a mouse model of systemic candidiasis, determined by use of a DPP3 knockout mutant of Candida albicans. Infect Immun 75: 1609-1618. Rajagopal, S., Eis, N., Bhattacharya, M., and Nickerson, K.W. (2003) Membrane-derived oligosaccharides (MDOs) are essential for sodium dodecyl sulfate resistance in Escherichia coli. FEMS Microbiol Lett 223: 25-31. Ramage, G., Saville, S.P., Wickes, B.L., and Lopez-Ribot, J.L. (2002) Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule. Appl Environ Microbiol 68: 5459-5463.

48 Rossignol, T., Logue, M.E., Reynolds, K., Grenon, M., Lowndes, N.F., and Butler, G. (2007) Transcriptional response of Candida parapsilosis following exposure to farnesol. Antimicrob Agents Chemother 51: 2304-2312. Saidi, S., Luitaud, C., and Rouabhia, M. (2006) In vitro synergistic effect of farnesol and human gingival cells against Candida albicans. Yeast 23: 673-687. Scheper, M.A., Shirtliff, M.E., Meiller, T.F., Peters, B.M., and Jabra-Rizk, M.A. (2008) Farnesol, a fungal quorum-sensing molecule triggers apoptosis in human oral squamous carcinoma cells. Neoplasia 10: 954-963. Semighini, C.P., Hornby, J.M., Dumitru, R., Nickerson, K.W., and Harris, S.D. (2006) Farnesol-induced apoptosis in Aspergillus nidulans reveals a possible mechanism for antagonistic interactions between fungi. Mol. Microbiol. 59: 753-764. Semighini, C.P., Murray, N., and Harris, S.D. (2008) Inhibition of Fusarium graminearum growth and development by farnesol. FEMS Microbiol Lett 279: 259-264. Shirtliff, M.E., Krom, B.P., Meijering, R.A., Peters, B.M., Zhu, J., Scheper, M.A., Harris, M.L., and Jabra-Rizk, M.A. (2009) Farnesol-induced apoptosis in Candida albicans. Antimicrob Agents Chemother 53: 2392-2401. Togashi, N., Inoue, Y., Hamashima, H., and Takano, A. (2008) Effects of two terpene alcohols on the antibacterial activity and the mode of action of farnesol against Staphylococcus aureus. Molecules 13: 3069-3076. Uppuluri, P., Mekala, S., and Chaffin, W.L. (2007) Farnesol-mediated inhibition of Candida albicans yeast growth and rescue by a diacylglycerol analogue. Yeast 24: 681-693.

49 Figure Legends Fig. 2-1. Effect of farnesol on C. albicans cell growth. A) stationary phase inoculum, B) exponential phase inoculum. Cells were grown in duplicate on at least two separate occasions in defined GPP medium with indicated levels of farnesol (see legend in B) at 30C in 96-well plates, and OD600 values were recorded on an automated plate reader (Molecular Devices, Sunnyvale, CA). Note the different y-axis scales in graphs A and B.

Fig. 2-2. Effect of farnesol on C. albicans cell death. Percent death was determined by methylene blue staining (Gibson et al., 2009). Cells were incubated in either PBS (A, C) or GPP (B, D) with the indicated levels of farnesol in 96-well plates at 30C. Incubations were initiated with either exponential (A, B) or stationary phase (C, D) inocula.

Fig. 2-3. Toxicity of farnesol to exponential cultures of C. albicans. One culture (A, B) was subdivided (in six) when it had reached exponential phase (OD600 = 0.5) and the other (C, D) was subdivided when it had reached stationary phase (OD600 = 4.0). Cultures were not washed prior to subdivision and farnesol addition. Cultures containing the indicated levels of farnesol were shaken at 30C and 250 rpm for 4 hrs and at the indicated times cell growth (A, C) and % dead cells (B, D) were determined. All cultures were in glass flasks in GPP both before and after subdivision.

50 Figure 2-1

51 Figure 2-2

52 Figure 2-3

53

CHAPTER 3 Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentousgrowth induction

Reference: Kebaara, B.W., Langford, M.L., Navarathna, D.H., Dumitru, R., Nickerson, K.W., and Atkin, A.L. (2008) Candida albicans Tup1 is involved in farnesol-mediated

inhibition of filamentous-growth induction. Eukaryot. Cell. 7: 980-7.

54 Abstract Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with

virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress expression of hyphal specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1regulated, hyphal-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Further, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with Tup1 being a crucial component of the response to farnesol in C. albicans.

55 Introduction Candida albicans is the most commonly isolated opportunistic fungal pathogen in humans. C. albicans is part of the normal flora, and it resides in the gastrointestinal and genitourinary tracts, as well as on the skin. However, C. albicans is capable of causing a wide range of disease, from mild mucosal infections to life-threatening systemic infections termed candidemia (Jarvis and Martone, 1992). Vulnerable patients include AIDS patients, patients undergoing chemotherapy, and organ transplant patients (Jarvis and Martone, 1992). The annual cost of treating candidiasis in the United States was estimated to be one billion dollars, and the mortality rates for patients with candidiasis are 30-50% even with antifungal treatment (Kullberg and Filler, 2002), indicating a need for new antifungal drugs. The ability of C. albicans to cause disease has been strongly linked to its conversion between two distinct morphological forms: yeast and filaments. Recently, our research has focused on farnesol, the first quorum sensing molecule (QSM) discovered in a eukaryote (Hornby et al., 2001). Farnesol is a virulence factor

(Navarathna et al., 2007a) that is excreted continuously by C. albicans (Hornby et al., 2001), and when it accumulates beyond a threshold level it blocks the yeast to filament conversion (Hornby et al., 2001). Stationary phase cultures of C. albicans have accumulated 2-4 M farnesol (Hornby et al., 2001) and the IC50 value for blocking germ tube formation (GTF) in an N-acetylglucosamine stimulated assay is ca. 1-2 M (E, E) farnesol (Hornby et al., 2001; Mosel et al., 2005; Shchepin et al., 2003) and, consequently, these farnesol production levels are physiologically relevant. Other roles described for farnesol include biofilm inhibition (Ramage et al., 2002), protection from

56 oxidative stress (Deveau et al., 2010; Westwater et al., 2005), and induction of apoptosis in another fungus, Aspergillus nidulans (Semighini et al., 2006). While many

phenotypic effects produced by farnesol have been described, little is understood about farnesols mode of action. In addition to farnesol, C. albicans yeast and filamentous growth is controlled by an assortment of signaling pathways (Berman and Sudbery, 2002; Dhillon et al., 2003). The yeast to filamentous conversion is activated by many pathways, including: Components of the CEK1 MAP kinase pathway, Ras/cAMP-dependent pathway, Calcium/Calmodulin signaling pathway, Rim101-independent pathway, and Chk1 twocomponent signal transduction pathway. Although each has been implicated in filamentation (Csank et al., 1998; Kruppa et al., 2004; Ramon et al., 1999), these pathways show some degree of specialization in that they respond to different environmental inducers. Activation and inhibition of filament development is largely accomplished through changes in gene expression mediated by transcription activators and repressors. Efg1 is a major transcription regulator of filamentous growth and is a central control point for many signaling pathways involved in filamentation (Eckert et al., 2007). Efg1 also regulates the expression of multiple genes including those involved in virulence (Eckert et al., 2007; Kumamoto and Vinces, 2005a). Mechanisms have also been identified that block filament development with transcriptional repression by Tup1 playing a key role (Braun and Johnson, 1997; Braun and Johnson, 2000). Farnesol is able to block filamentous growth induced by environmental signals for most, and possibly all of the signaling pathways activating filament development. These signals include: 10% serum, 10 mM L-proline, 2.5 mM N-acetylglucosamine, or the

57 combination of N-acetylglucosamine and L-proline, all at 37oC (Hornby et al., 2001). Thus, farnesol may individually block each of the morphogenic signaling pathways and/or act at a common control point in morphogenesis. Tup1 repression of filament specific genes is an attractive candidate for a common control point that may be regulated by farnesol (Kadosh and Johnson, 2005). The C. albicans Tup1 protein is a transcription regulator that plays two key roles in the cell: 1) regulation of phase switching, and 2) inhibition of filamentous growth. Tup1 interacts with either Ssn6 or Tcc1 corepressor proteins. This complex functions with DNA binding proteins to repress gene expression (Kaneko et al., 2006; Sprague et al., 2000). At least three DNA-binding proteins have been identified that function with Tup1: Nrg1 (homologous to Saccharomyces cerevisiae Nrg1p), Rfg1 (homologous to S. cerevisiae Rox1p), and Mig1 (homologous to S. cerevisiae Mig1p). Homozygous tup1 mutants are unable to grow as yeast and instead remain locked in the filamentous form in all media tested (Braun and Johnson, 1997). Deletion of TUP1 results in the up-

regulation of approximately one-third of C. albicans genes (Murad et al., 2001a; Murad et al., 2001b) and these mutants are also avirulent in a murine model of infection. Activation of Tup1 transcription repressor complexes results in the repression of filament-specific gene expression (Braun and Johnson, 1997; Braun and Johnson, 2000; Murad et al., 2001a; Murad et al., 2001b). Here, we tested the hypothesis that the C. albicans response to farnesol involves Tup1. The morphological response to farnesol was tested in wild-type, tup1/tup1, tup1/TUP1, nrg1/nrg1, and rfg1/rfg1 strains to assess the requirement for these genes in the farnesol response. The gene expression pattern for MIG1 was not determined because

58 Mig1 protein does not play a role in filamentous growth of C. albicans (Murad et al., 2001a). The gene expression patterns of TUP1, NRG1, RFG1, and EFG1, as well as genes under their control were examined in the presence or absence of farnesol by quantitative northern and western analyses. Finally, we compared farnesol production levels in tup1, nrg1, and rfg1 homozygous mutants relative to wild type cells.

59 Methods Strains and media Candida albicans SC5314 is an independent clinical isolate and the reference strain for the Candida genome sequence. C. albicans strains CAF-2

(ura3:imm434/URA3) and CAI-4 (ura3::imm434/ura3::imm434) are derived from SC5314 by gene replacement . Strains BCa2-9 (tup1/tup1, in CAI-4; (Braun and

Johnson, 1997), BCa2-10 (tup1/tup1, frameshift disruption fragment in CAI-4; (Braun and Johnson, 1997), DU152 (nrg1/nrg1, in CAI-4; (Braun and Johnson, 1997), DU129 (rfg1/rfg1, in CAI-4; (Backen et al., 2000; Kadosh and Johnson, 2001), BCa05 that expresses TUP1 ectopically (tup1/tup1, MAL3:p455, in CAI-4; (Braun and Johnson, 1997) and BCa2-3 (TUP1/tup1, in CAI-4; (Braun and Johnson, 1997) were obtained from Alexander Johnson, University of California, San Francisco, CA. Strain MEN was provided by Richard Cannon, University of Otago, Dunedin, New Zealand. Resting cells were obtained by growing cells in modified glucose salts biotin media (mGSB) overnight, washing three times with 50 mM phosphate pH 6.5, resuspended in 10 ml of 50 mM phosphate, and stored at 4C to be used within a month. The defined glucose-salts medium GPP pH 4.8 contained (per liter distilled water): glucose, 20g; L-proline, 1.15g; NaH2PO4, 3.2g; KH2PO4, 4g; MgSO47H20, 0.5g; CuSO45H20, 1mg; ZnSO47H20, 1mg; MnCl2, 1mg; FeSO4, 1 mg; biotin, 20 g; pyridoxineHCl, 200 g; thiamineHCl, 200 g,. The glucose (20% w/v) and L-proline (100 mM) were autoclaved separately and added aseptically, as were the filter-sterilized vitamins (Kulkarni and Nickerson, 1981). Modified GPP (mGPP) also contained 2.5 mM N-acetylglucosamine (Hornby et al., 2001). GPP pH 6.8 contained 3.2g/L Na2HPO4

60 instead of NaH2PO4. For maltose phosphate proline media (MPP), filter-sterilized

maltose replaced the glucose. Cornmeal agar (Difco, Detroit, MI) was also used. Solid media included 1.5% (w/v) agar. All media for CAI-4 included uridine at 40 g/ml (Backen et al., 2000). Microscopy Differential interference contrast (DIC) images were photographed with an Olympus BX51 microscope and colony morphology images were photographed with an Olympus SZX12 microscope. Quantitative Northern blot analysis To measure mRNA accumulations, SC5314 resting cells were inoculated in mGPP to an OD600 of 0.5-0.6 and allowed to equilibrate at 37C for 5 minutes whereupon 20 M farnesol was added to half of the flasks. Cells were grown at 37C for 0, 20, 40, 60, and 80 minutes until the cells were harvested and total RNA extracted by the hot phenol method (Backen et al., 2000; Kebaara et al., 2003). Equal amounts of RNA (15g) were resolved on 1.0 % agarose-formaldehyde gels and the RNA was transferred to GeneScreen Plus (NENTM Life Science Products, Inc., Boston, MA) using the capillary blot transfer protocol recommended by the manufacturer. The Northern blots were probed with radiolabeled DNA probes. The probe DNAs used for synthesis were prepared by PCR using MEN genomic DNA. The probes were labeled with
32

P-dCTP (GE Health

Sciences, Piscataway, NJ) using an oligolabeling kit, RadPrime DNA labeling system following the protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Northern blots were PhosphorImaged using a Storm (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and quantified using ImageQuant software (Molecular Dynamics version

61 5.0, Sunnyvale, CA). mRNA abundance measurements were done using a minimum of three independent Northern blots. Western blot analysis Western blots were prepared as previously described (Atkin et al., 1995) and Tup1 and Act1 proteins detected with the Supersignal West Pico Chemiluminescent Substrate using the manufacturers protocol (Pierce, Rockford, IL) with the exception that blocking was done with 5% nonfat dry milk. Rabbit polyclonal antibodies against Tup1 were previously described (Inglis and Johnson, 2002). Mouse monoclonal Anti-Act1 antibodies and horseradish peroxidase (HRP) labeled ant-rabbit IgG antibodies were from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). HRP labeled anti-mouse antibody was from Perkin-Elmer (Boston, MA). Analysis of farnesol levels Extracellular farnesol was extracted from cell free-supernatants of cultures grown in mGPP at 30o and analyzed by GC/MS as described (Hornby et al., 2001).

62 Results tup1/tup1 and nrg1/nrg1 mutants lack a morphological response to farnesol while the rfg1/rfg1 mutant responds to farnesol The juxtaposition of farnesols ability to inhibit differentiation, and the role of Tup1 as a transcription repressor for filamentation genes suggests that farnesol could function by activating Tup1 and/or one of its co-regulators, Nrg1 and Rfg1. Consequently, we examined the effect of farnesol on the morphology of null mutants lacking TUP1, NRG1, and RFG1. As a control, the wild type C. albicans SC5314 in filament-inducing media grew as yeasts in the presence of 20 M farnesol and as filaments in media lacking farnesol, demonstrating a positive response to farnesol (Fig. 1). The rfg1/rfg1 mutant responded to 20 M farnesol in a similar manner to SC5314 (Fig. 1). Unlike SC5314 and rfg1/rfg1, the tup1/tup1 and nrg1/nrg1 mutants lacked a detectable response to farnesol and remained filamentous in the presence of 20 M farnesol (Fig. 1). The filamentous-only cell morphology is the expected phenotype for these known mutants (Braun and Johnson, 1997; Braun et al., 2001; Khalaf and Zitomer, 2001; Murad et al., 2001a; Murad et al., 2001b). However, in this regard, tup1/tup1 and nrg1/nrg1 mutants differ from the great majority of filamentous-only mutants recovered from a previous study, 96% of which reverted to a smooth colony (yeast) morphology on YM agar plates with 50 M farnesol (Jensen et al., 2006). For the tup1/tup1 mutant, the lack of response to farnesol was specific for loss of Tup1 because we found ectopic expression of TUP1 (Braun and Johnson, 1997) restores the ability to respond to farnesol (data not shown).

63

TUP1 mRNA levels increase in the presence of farnesol while RFG1 and NRG1 mRNA levels were not affected by farnesol We analyzed the effect of farnesol on TUP1 mRNA levels over time in C. albicans SC5314 cells which had been induced to differentiate from yeasts to filaments by growth at 37C in mGPP. We previously showed (Mosel et al., 2005) that, in these conditions, the first germ tubes appeared at 30 min and the process was complete by 110 min. Further, farnesol no longer blocked germ tube formation when added 60-90 min after inoculation (Kulkarni and Nickerson, 1981). Here our analysis was designed to

evaluate changes in TUP1 mRNA just before germ tube formation when the cells were still responsive to farnesol. Filamentation was induced by transferring resting cells into mGPP (pH 4.8) at 37C in the presence and absence of 20 M farnesol and mRNA levels were determined at 0, 20, 40, 60 and 80 min following induction. In all experiments, the TUP1 mRNA levels decreased over the first 20 min and then increased (Fig. 2A). This pattern is consistent with the single time point results of Toyoda et al. (Toyoda et al., 2004) who showed that TUP1 mRNA levels increased slightly at 180 minutes after induction of filamentation. In the presence of farnesol, we found that TUP1 mRNA consistently increased 2.50.6 (n=4) fold from 20 to 60 min. Importantly, this is the time period just prior to that at which the cells become committed and are no longer responsive to farnesol (Mosel et al., 2005). In contrast, in the absence of farnesol, there was very little increase (1.40.3, n=4) in TUP1 mRNA levels from 20 to 60 min (Fig 2A). Thus, farnesol (20 M) causes a consistent increase in TUP1 mRNA levels during the precise time period when it blocks differentiation from yeasts to filaments. This

64 increase of 2.5-fold in TUP1 mRNA corresponded to an increase in SC5314 Tup1 protein levels at 60 min following induction (Fig.3). Tup1 protein in SC5324 was increased in all three replicate experiments by an average of 2.5-fold. Since Tup1 functions with DNA binding proteins such as Rfg1 or Nrg1, and in C. albicans strain JCM9061 NRG1 mRNA levels decreased during filamentation (Toyoda et al., 2004), we tested the effect of farnesol on RFG1 and NRG1 mRNA levels during differentiation from yeasts to filaments. Like TUP1, RFG1 mRNA levels initially

decreased and then increased (data not shown). However, unlike TUP1, the timing and magnitude of the changes were similar in the presence and absence of farnesol (Fig. 4, data not shown). Under the same conditions, NRG1 mRNA levels did not change during development and they too were the same in the presence and absence of farnesol (Fig. 4). Thus, we conclude that farnesol does not affect RFG1 or NRG1 mRNA levels.

Expression of the Tup1-regulated filamentous genes HWP1 and RBT1 is inhibited by farnesol To test whether the increased TUP1 expression in the presence of farnesol was biologically significant, we examined the expression of two Tup1-regulated genes, HWP1 and RBT1 [Fig. 2B and C, (De Groot et al., 2003)]. In the absence of farnesol, the HWP1 and RBT1 transcripts were undetectable at time 0 but they were strongly expressed from 40 to 80 minutes (Fig. 2B and C). Farnesol delays and dramatically reduces the

magnitude of HWP1 and RBT1 mRNA expression (Fig. 2B and C). At 80 minutes, HWP1 and RBT1 levels were 30- and 7.6-fold lower, respectively, in farnesol treated than untreated cells. Similar results were observed by Davis-Hanna et al. (Davis-Hanna et al.,

65 2008) for HWP1 mRNA 2 hours after treatment with 75 M farnesol. Thus there is a strong correlation between elevated TUP1 expression in response to farnesol and the expression of Tup1-regulated genes.

EFG1 mRNA levels remain unaffected by farnesol Efg1 is a transcription regulator for genes required for filamentation. EFG1 mRNA levels are down regulated at the initiation of filament development and then increase as filament formation progresses (Tebarth et al., 2003). HWP1 and RBT1 are activated by Efg1 during filamentation. Therefore, we tested whether farnesol also

affects EFG1 mRNA levels (Fig. 4). The EFG1 mRNA levels were high at time 0, decreased to a minimum at 20 minutes, and then increased steadily throughout the remaining time (data not shown). However, farnesol had no influence on EFG1 mRNA levels since the timing and magnitude of the changes were similar in the presence and absence of farnesol (Fig.4 and data not shown).

Farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Braun and Johnson (Braun and Johnson, 1997) showed that BCa2-3, a TUP1/tup1 heterozygote, is haploinsufficient in that these cells develop a higher proportion of filaments compared to wild-type cells on most media (Braun and Johnson, 1997). Presumably these cells do not make enough Tup1 to compensate for the reduced gene copy number. We hypothesized that farnesol might suppress this phenotype because it increases TUP1 expression 2.5-fold in SC5314 and 4.2 fold in TUP1/tup1 (Fig. 3). This increase should restore Tup1 to roughly wild type levels. To test this hypothesis, we

66 examined the effect of farnesol on C. albicans BCa2-3 on cornmeal agar plus Tween 80 under a coverslip for 25 hours at 25C. In these conditions, the TUP1/tup1 mutant was more filamentous than wild-type colonies but less filamentous than the tup1/tup1 mutant (BCa2-10; 4 and Table 2). As a control, we showed the TUP1/tup1 mutant responds to farnesol because, although it forms filamentous cells when grown in mGPP, the addition of 20 M farnesol results in growth as yeasts (Fig. 1), and Tup1 protein levels were ca. 4.2-fold higher in the TUP1/tup1 mutant treated with farnesol. In contrast to the haploinsufficient phenotype observed in the absence of farnesol, in the presence of farnesol the TUP1/tup1 mutant looked identical to wild type C. albicans (Table 2). Thus farnesol suppresses the haploinsufficiency phenotype of the TUP1/tup1 heterozygote.

tup1/tup1 and nrg1/nrg1 mutants produce excess farnesol. Jensen et al (Jensen et al., 2006) tested the farnesol production levels for several filamentous-only mutants. A subset of these mutants produced levels of farnesol

significantly higher than wild type strains. This overproduction suggests the ability to respond to farnesol may be linked to regulation of farnesol production. Here we tested farnesol production levels in CAI-4, CAF-2, tup1/tup1, nrg1/nrg1, and rfg1/rfg1 strains. Farnesol production levels were dramatically increased in the tup1/tup1 and nrg1/nrg1 mutants (Table 1) that were unable to respond to farnesol (Fig. 1). The tup1/tup1 and nrg1/nrg1 mutants produced ca. 17- and 19-fold more farnesol, respectively, than did CAF-2 and CAI-4. In contrast, the farnesol responsive rfg1/rfg1 mutant only produced ca. 2.6-fold more farnesol than the wild-type strains (Table 1). Thus, the two mutants

67 that are unable to respond to farnesol (tup1/tup1 and nrg1/nrg1) produced much higher levels of farnesol than did strains that do respond to farnesol.

tup1/tup1 overproduction of farnesol inhibits SC5314 filamentation. We tested the biological significance of farnesol overproduction by sequentially plating tup1/tup1 and SC5314 next to one another and observing the resultant colony morphologies. When SC5314 was plated and followed one day later by another streak with SC5314, a small area of filament inhibition was observed (Fig. 5A). In contrast, when tup1/tup1 was plated first, followed by SC5314, a much larger area of filament inhibition was observed (Fig. 5B). These results are consistent with the tup1/tup1 overproduction of farnesol. As controls, whenever tup1/tup1 was plated second, no filament inhibition was observed (Fig. 5, C and D).

68 Discussion C. albicans responds to farnesol, in part, by changing gene expression (Cao et al., 2005; Enjalbert and Whiteway, 2005). We hypothesize that some of these changes are mediated by changes in the activity of the signaling pathways regulating morphogenesis. Here we show that the tup1/tup1 and nrg1/nrg1 null mutants are strictly filamentous and the cells remain filamentous in the presence of added farnesol (Fig. 1). In these cases the total farnesol levels are actually much higher than the added farnesol because the mutants themselves produce elevated levels of farnesol (Table 1, see below). Further, Tup1 mRNA and protein levels increased in the presence of farnesol while two Tup1-regulated genes, HWP1 and RBT1 mRNA levels decrease (Fig. 2, and 3). Importantly, the timing of this increase (40-60 min, Fig. 2) corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation (Mosel et al., 2005). Finally, we believe that Tup1 is part of the farnesol response pathway because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 strain (Table 2). Cell synchrony, farnesol concentration, and timing were all important considerations for our experimental design. Previous work examining farnesol-

dependent changes in the global transcription profiles of developing biofilms (Cao et al., 2005; Enjalbert and Whiteway, 2005) and during resumption of growth following stationary phase (Enjalbert and Whiteway, 2005) were done with mixed cell populations that differed in their ability to respond to farnesol. Further, the effect of farnesol addition on the global gene expression during biofilm formation was determined at a single time point, 24 hours after addition of farnesol (Cao et al., 2005). This point is significant because such a study could only measure stable long-term farnesol-dependent changes in

69 gene expression. Timing is also important because of the commitment phenomenon. This is the point at which a switch in the environmental stimulus no longer causes the expected switch in morphology (Chaffin and Wheeler, 1981; Mitchell and Soll, 1979; Mosel et al., 2005). It is relevant to farnesols mode of action because, while farnesol blocks the yeast to filament switch, it does not block the elongation of preexisting filaments (Mosel et al., 2005). Thus for our experiments, we added farnesol at time zero in order to avoid commitment to filamentous growth, and we harvested cells at 20 minute increments to observe changes in transcript levels during the early stages of the farnesol response (Mosel et al., 2005). We also achieved a synchronous cell population by starting with resting cells and inoculating them in mGPP; in these conditions we routinely get 95-100% filaments within 3-4 hrs. Exposing a synchronized cell population to

farnesol allowed us to detect subtle and consistent changes in transcript abundance. Small changes in expression of a transcription regulator can have profound effects on the genes it regulates. For example, we have shown that nonsense-mediated mRNA decay (NMD) in S. cerevisiae regulates accumulation of the mRNA for Adr1, a transcription regulator of the genes responsible for making acetyl CoA and NADH from nonfermentable substrates. In particular, the respiratory impairment seen in NMD

mutants is due, in part, to overexpression of Adr1 (Taylor et al., 2005). The change in ADR1 mRNA levels is small (2.6-fold), but sufficient to affect expression of Adr1regulated genes. Thus even though the change in Tup1 expression is relatively small it can have a profound effect on expression of the genes it regulates. Two Tup1 co-regulators, encoded by NRG1 and RFG1, were unaffected by farnesol at the mRNA level (Fig. 4). In this regard, it is reasonable that farnesol regulates

70 only one part of the complex, i.e. that farnesol elevates TUP1 mRNA but not NRG1 or RFG1 mRNA. By analogy, for Ca2+ and calmodulin where only the Ca2+-calmodulin complex is active (Soto et al., 2004a), fungi have the calmodulin in excess and regulate the activity of the complex by regulating the availability (Muthukumar et al., 1987). Because the tup1/tup1 and nrg1/nrg1 mutants did not respond to farnesol, it suggests that farnesol acts through a pathway requiring Tup1 and Nrg1. The rfg1/rfg1 mutant responded to farnesol, indicating that the genes regulated by Rfg1 are not required for the response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants of cytoplasmic Ca2+

overproduced farnesol while the rfg1/rfg1 mutant produced only slightly elevated farnesol (Table 1). This tup1/tup1 mutant overproduction is biologically significant because the excess farnesol produced by the tup1/tup1 mutant inhibits filamentation of wild-type C. albicans grown on the same plate (Fig. 5). The juxtaposition between farnesol non-responsive mutants and the overproduction of farnesol implies that a farnesol-Tup1 feedback loop may exist, and that Nrg1 may work in concert with Tup1 to negatively regulate farnesol synthesis. This regulation may be direct or indirect. The enzyme responsible for ca. 90% of farnesol synthesis is Dpp3 (Navarathna et al., 2007a). DPP3 mRNA levels were not significantly elevated in the whole genome profiles of tup1/tup1 or nrg1/nrg1 mutants (Kadosh and Johnson, 2005); however, DPP3 does have a putative Nrg1 binding site in its promoter region. The increased TUP1 expression we observed for farnesol blockage of filament development (Fig. 2A) is smaller than that reported for farnesol blockage of biofilm development, ca 6.6-fold as determined with DNA arrays (Cao et al., 2005). The

71 difference in the response intensity may reflect filamentous vs. biofilm growth conditions as well as the fact that Cao et al. (Cao et al., 2005) used one time point 24 hours after farnesol addition. Efg1 is a transcriptional factor that activates hyphal gene expression including HWP1 and RBT1. EFG1 mRNA levels are regulated during filamentation, but they were not affected by farnesol since the timing and magnitude of the changes were similar in the presence and absence of farnesol (Fig. 4). These results are consistent with those of Soto et al (Soto et al., 2004b) who also found no change in EFG1 mRNA levels at a single time point with added farnesol (Soto et al., 2004b). Together with our results, this suggests that farnesol does not regulate EFG1 mRNA levels but, at this time we can not exclude the possibility that post translational regulation of Efg1 is affected by farnesol. Two other farnesol-related findings regarding filamentous growth can be accommodated in a Tup1-dependent model because they are downstream from Tup1. Soto et al (Soto et al., 2004b) suggested that farnesol acts by causing decreased CPH1 and HST7 mRNA levels. CPH1 is a transcription factor that regulates filamentous growth and HST7 is a MAP kinase kinase involved in filamentous growth. Both are downregulated by Tup1 and thus their downregulation by farnesol (Soto et al., 2004b) is consistent with a secondary effect of farnesol on Tup1. Additionally, Chk1, a histidine kinase shown to be required for the farnesol response (Kruppa et al., 2004), is also a Tup1 repressed gene; CHK1 was elevated 6.5-fold in the tup1/tup1 mutant (Kadosh and Johnson, 2005). Taken together, these findings indicate that Tup1 is involved in

mediating the C. albicans response to farnesol.

72 Acknowledgements We thank Alexander Johnson and Richard Cannon for providing us with C. albicans strains and the Tup1 antibody. We also thank Jessica A. Wiles for assisting with the germ tube assays and RNA work. This work was supported by grants from the National Science Foundation (MCB0110999), the University of Nebraska Tobacco Settlement Biomedical Research Enhancement Fund, and the Farnesol and Candida albicans Research Fund, University of Nebraska Foundation. Any opinions, findings, conclusions, or recommendations expressed in this report are ours, and do not necessarily reflect the views of the National Science Foundation.

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78 Shchepin, R., Hornby, J.M., Burger, E., Niessen, T., Dussault, P., and Nickerson, K.W. (2003) Quorum sensing in Candida albicans: probing farnesol's mode of action with 40 natural and synthetic farnesol analogs. Chem. Biol. 10: 743-750. Soto, T., Ueno, Y., Watanabe, T., Mikami, T., and Matsumoto, T. (2004a) Role of Ca2+/calmodulin signaling pathway on morphological development of Candida albicans. Biol. Pharm. Bull 27: 1281-1284. Soto, T., Watanabe, T., Mikami, T., and Matsumoto, T. (2004b) Farnesol, a morphogenetic autoregulatory substance in the dimorphic fungus Candida albicans, inhibits hyphae growth through suppression of a mitogen-activated protein kinase cascade. Biol Pharm Bull 27: 751-752. Sprague, E.R., Redd, M.J., Johnson, A.D., and Wolberger, C. (2000) Structure of the Cterminal domain of Tup1, a corepressor of transcription in yeast. EMBO J. 19: 3016-3027. Taylor, R., Kebaara, B.W., Nazarenus, T., Jones, A., Yamanaka, R., Uhrenholdt, R., Wendler, J.P., and Atkin, A.L. (2005) Gene set coregulated by the Saccharomyces cerevisiae nonsense-mediated mRNA decay pathway. Eukaryot Cell 4: 20662077. Tebarth, B., Doedt, T., Krishnamurthy, S., Weide, M., Monterola, F., Dominguez, A., and Ernst, J.F. (2003) Adaptation of the Efg1p morphogenetic pathway in Candida albicans by negative autoregulation and PKA-dependent repression of the EFG1 gene. J Mol Biol 329: 949-962.

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80 Figure Legends Figure 3-1. Response to farnesol by C. albicans in conditions that promote germ tube formation and hyphal growth. SC5314, CAI4, CAF2, rfg1/rfg (DU129), nrg1/nrg1

(DU152), tup1/tup1(BCa2-9), tup1/tup1 (BCa2-10) and TUP1/tup1 (BCa2-3) resting cells were inoculated to mGPP (pH 4.8) medium at 37C in the presence or absence of 20 M farnesol, and their cell morphology examined at 4 hours. Scale bar = 10m

Figure 3-2. TUP1 mRNA levels increased, while two Tup1-regulated genes, HWP1 and RBT1 were downregulated in the presence of farnesol. C. albicans SC5314 resting cells were inoculated into mGPP (pH 4.8) in the presence or absence of 20 M farnesol and incubated at 37C. Cells were then harvested at 0, 20, 40, 60 and 80 minutes post inoculation. Northern blots were prepared with total RNA from cells incubated both in the presence or absence of farnesol. Shown is a PhosphorImage of a representative Northern blot probed with radiolabeled TUP1 DNA (A), HWP1 DNA (B) and RBT1 DNA (C), and a plot of average mRNA levels from a minimum of three independent experiments. ACT1 mRNA levels were used as a loading control.

Figure 3-3. Tup1 protein levels are higher in the presence of farnesol. Total protein extracts were prepared from SC5314 and TUP1/tup1 (BCa2-3) resting cells inoculated to mGPP (pH 4.8) medium at 37C in the presence or absence of 20 M farnesol and incubated at 37C for 60 minutes. The average fold Tup1 protein accumulation for farnesol treated cells relative to untreated cells is shown. Act1 levels were used as a loading control.

81

Figure 3-4. Farnesol does not affect expression of RFG1 or NRG1, which encode DNA binding proteins that function with Tup1, or EFG1, which encodes a transcription activator of hypal-specific genes. Quantitative Northern blot analysis was used to

measure TUP1, NRG1, RFG1, and EFG1 mRNA levels in SC5314 at 60 min after inoculation of resting cells into conditions that promote germ tube formation and hyphal growth in the presence and absence of 20M farnesol. The results are an average of three independent experiments.

Figure 3-5.

Overproduction of farnesol by the tup1/tup1 mutant inhibits SC5314

filamentation. Resting cells were grown at 37 C for 24 hours on YPD agar plates to allow for farnesol accumulation in the agar (horizontal streak, C.albicans strain SC5314 (B, C), or tup1/tup1 (BCa2-10, A, D)). Subsequently, either SC5314 (A, B) or tup1/tup1(C, D) resting cells was plated (vertical streak) and incubated at 37 C for an additional 24 hours. Area above the two arrows (left panels A, B) are zones of filament inhibition (as evident by smooth morphology) resulting from the farnesol produced by the horizontally streaked strains. Filamentation gives the wrinkled colony morphology seen below the arrows. The pictures in the two white boxes have been magnified 2.5X so that the colony morphology can be seen more clearly (center panels A, B). Micrographs of individual cells from the two bracketed regions are shown in the right panels (A, B; scale bar = 10m). The cells from the smooth regions are mainly yeasts, while there is a much larger proportion of filaments in the wrinkled region.

82 Figure 3-1

83 Figure 3-2

84 Figure 3-3

85 Figure 3-4

86 Figure 3-5

87
Table 3-1. tup1/tup1 and nrg1/nrg1 null mutants do not respond to farnesol but overproduce farnesol. C. albicans strain (CAI-4) (CAF-2) tup1/tup1(BCa2-10) nrg1/nrg1 (DU152) rfg1/rfg1 (DU129) Farnesol responsea/ Positive Positive Negative Negative Positive Farnesol productionb/ 1.6 0.36 2.0 1.30 30.6 6.40 34.5 12.2 4.8 2.0 17 19 2.6 Fold increase in farnesolc/

a/

Farnesol responses on GPP agar with and without 20M farnesol incubated at 37oC for

48 hours. A positive response to farnesol indicates smooth colony morphology (yeast cells) in the presence of farnesol and rough colony morphology (filamentous cells) without added farnesol. A negative response to farnesol indicates rough colony

morphology in the presence and absence of farnesol.

b/ c/

Farnesol production (g/g dry weight of cells) was the average of three measurements Values based on fold increase over 1.8, the average value for CAI-4 and CAF-2 strains.

88 Table 3-2. Farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Cells were plated on a cornmeal agar plus Tween 80 plate under a coverslip and grown at 25C for 25 hours. C. albicans Strain Cell morphology at the colony periphery No Farnesola/ Wild type (SC5314) TUP1/tup1 (BCa2-3) tup1/tup1 (BCa10) Yeasts + few filaments Yeasts + filaments filaments 20 M Farnesol Yeast + few filaments Yeast + few filaments filaments

a/

The phenotype for these strains grown on a cornmeal agar plus Tween 80 plate under a

coverslip without farnesol was also previously reported (Braun and Johnson, 1997).

89

CHAPTER 4 A novel role for Czf1 in farnesol tolerance and the morphological response to farnesol in Candida albicans

90 Abstract Candida albicans is the primary cause of hospital acquired fungal infections in the United States. The quorum sensing phenomenon in Candida albicans is a process that inhibits the transition to filamentous growth and may be directly related to its ability to cause both mucosal and systemic disease. The Ras1-cyclic AMP signal transduction pathway is a proposed target for farnesol inhibition, resulting in filamentous growth obstruction, but a clear understanding of the downstream effectors and signaling networks vital for the morphological farnesol response have yet to be unraveled. To address this issue, we screened a mutant library for farnesol resistant mutants; among the resistant mutants was a czf1/czf1 mutant. Here, we demonstrate for the first time the capability of C. albicans to respond to farnesol in embedded conditions, and in these and liquid assays, Czf1 was required for a wild-type farnesol response. The CZF1 mRNA transcript levels in response to farnesol, farnesol toxicity, and farnesol production levels were also examined. Finally, CZF1 ectopic expression restored farnesol response in strains lacking Czf1, highlighting a new role for Czf1 as a critical downstream effector of the morphological response to farnesol in C. albicans.

91 Introduction Candida albicans is an opportunistic pathogen that is part of the normal flora but can cause mild to severe opportunistic infections in immunocompromised patients. A polymorphic fungus, C. albicans can switch between yeast and filamentous forms of growth, and this transition is necessary for causing disease in a mouse model of infection (Lo et al., 1997; Saville et al., 2003). Two additional growth morphologies in C.

albicans include mating-competent opaque cells and structures with unknown function called chlamydospores. All of these cell types are affected by the quorum sensing molecule, farnesol, highlighting its influential role in C. albicans morphology. Farnesol was the first eukaryotic quorum sensing molecule identified and was initially shown to block the transition from yeast to filaments (Hornby et al., 2001). Subsequent studies identified farnesol as having an inhibitory role in biofilm formation (Ramage et al., 2002) and a protective role against oxidative stress (Deveau et al., 2010; Westwater et al., 2005). In addition, very high levels of farnesol can increase

chlamydospore formation (Martin et al., 2005), and low levels of farnesol induces necrosis in opaque cells when oxygen is present, thereby regulating opaque cell stability (Dumitru et al., 2007). White cells can also be killed by farnesol in the right

environmental conditions; log phase cells that are energy-deprived are particularly sensitive, while stationary phase cells in growth medium are quite farnesol tolerant (Langford et al., 2010; Shirtliff et al., 2009; Uppuluri et al., 2007). Given its important role in physiology, it comes as no surprise that in vivo studies provide physiological relevance for farnesol signaling during infection and suggest farnesol plays distinct roles at different sites of infection. For example, farnesol is a virulence factor in a

92 disseminated mouse model of infection (Navarathna et al., 2007a), yet it protects mice from oral candidiasis (Hisajima et al., 2008). These results highlight the need for a complete understanding of the signaling response induced by farnesol in C. albicans. Factors identified that play a role in the C. albicans farnesol response include Tup1/Nrg1 (Kebaara et al., 2008), Chk1 (Kruppa et al., 2004), the Hog1 MAP kinase pathway (Smith et al., 2004), the Cek1 MAP kinase pathway (Roman et al., 2009), and the Ras1-cyclic AMP (cAMP) signaling pathway (Davis-Hanna et al., 2008). In this paper, we have identified Czf1 (C. albicans zinc finger) as an important factor in the response to farnesol by screening a mutant library for mutants with a defective farnesol response. Known roles of Czf1 include induction of contact-induced filamentous growth (Brown et al., 1999), biofilm formation (Stichternoth and Ernst, 2009), and white to opaque cell switching (Vinces and Kumamoto, 2007). It contains an unusually large 5 untranslated region (UTR) of approximately 2 kb (Vinces et al., 2006), and can negatively regulate its own mRNA expression (Vinces et al., 2006). Czf1 also has ties to the cAMP pathway, as a transcription factor downstream of cAMP signaling, Efg1, can bind the promoter of CZF1 and tightly regulate its expression, since no CZF1 mRNA was detected in an efg1/ efg1 background (Vinces et al., 2006). This positive regulation by Efg1 is intriguing because Czf1 and Efg1 appear to play opposing roles in the cell with respect to morphology (Giusani et al., 2002; Noffz et al., 2008). Of note, regulation between Efg1 and Czf1 may also occur at the protein level as well since these two proteins interact during a yeast two hybrid assay (Giusani et al., 2002; Noffz et al., 2008). Here we have defined a new role for Czf1 in the quorum sensing response of C. albicans. We show that Czf1 is not only necessary for a wild type morphological

93 response to farnesol in a variety of conditions, but in combination with Efg1, is required for farnesol tolerance as well. Our data suggest a vital downstream role for Czf1 in the signaling cascade elicited by farnesol in C. albicans.

94 Methods Strains and media Candida albicans strains and plasmids are listed in Table 1. The defined medium, modified glucose phosphate proline (mGPP) pH 6.8 was prepared as described in Kebaara et al (Kebaara et al., 2008) with 2.5 mM N-acetylglucosamine (GlcNAc) and uridine (40 g/ml). Yeast peptone dextrose (YPD) medium contained 1% yeast extract, 0.5% peptone, and 2% dextrose, and solid medium included 2% agar. Resting cells were prepared as in Kebaara et al (Kebaara et al., 2008) with modifications: single colonies were grown in 25 mL YPD broth at 30C (unless otherwise noted) for 22-24h to reach stationary phase, washed three times with 50 mM potassium phosphate buffer (pH6.8), resuspended in potassium phosphate buffer, and stored at 4C overnight before use. For liquid farnesol response assays, resting cells were inoculated at 106 cells/mL in mGPP or mSPP (2% sucrose replacing glucose) broth with indicated farnesol concentrations. Cells were then grown at 37C, shaking in glass flasks for the indicated times. Trans-trans farnesol (Sigma, St. Louis, MO) was stored under nitrogen and freshly prepared as a 100 mM stock solution in methanol for each experiment. Mutant library screen Mutants were obtained in 96-well plates and plated on mGPP (with 40 g/ml uridine, arginine, and histidine added) agar plates containing 0, 10, or 50 M farnesol. Plates were incubated at 37C for 2 days before colony morphology was assessed and compared to the parental strain, BWP17 for farnesol response. Wrinkled/hairy colony

morphologies were considered to be composed primarily of filaments and smooth colony morphologies were considered to be mostly yeast cells. A positive response to farnesol

95 was viewed as filamentous colonies without farnesol and smooth colonies in the presence of farnesol. Farnesol resistant mutants maintained a filamentous morphology, even in the presence of 50 M farnesol. Embedded cell growth Embedded media were prepared by mixing 104 cells/mL in 30 ml GPP (no GlcNAc added) or SPP molten agar (cooled to 50C) with appropriate concentrations of farnesol and plated. Embedded plates were incubated at 37C for 12 or 17 hours as indicated. Only colonies beneath the agar surface were examined. Microscopy and cell death determination Cellular morphology during the germ tube assays was determined using a Zeiss Stemi 2000-C light microscope. Embedded micrographs were taken using a custom MVI

TDM400 tetrad dissecting microscope and Sony Cybershot camera. DIC micrographs were taken using an Olympus BX51 microscope and a Photometrics CoolSnap HQ CCD camera. Cell death was determined by methylene blue staining as described by Gibson et al (Gibson et al., 2009). DNA analysis and transformation To create strain AAC2, CKY283 was plated on 5-fluoroorotic acid (5-FOA) containing media to select for ura- mutants. AAC2 was subsequently transformed with BsgI-

digested pDB212 to create strain AAC6. Transformations were performed by the lithium acetate method and transformants were selected on media lacking uridine (Gietz and Woods, 2002). Newly created strains were confirmed by PCR and Southern blot analysis (data not shown); restriction digestion and Southern blotting were performed as described

96 in the GeneScreen Plus Hybridization Transfer and Detection Protocols (DuPont NEN Research Products, Boston, MA). Quantitative real- time PCR Resting cells were inoculated into 75 mL mGPP broth at 5 x 106 cells/mL, and 0, 50, or 100 M farnesol was added to each flask. Cells were incubated at 37C and harvested at 40, 60, or 80 minutes. For harvesting cells, cultures were passed through glass fiber filters and cells were scraped off the filters to reduce the loss of filamentous cells during centrifugation. Data shown are from a representative independent experiment performed in triplicate. Extraction of mRNA was performed with the RiboPure Yeast Kit (Applied Biosystems, Foster City, CA), and mRNA integrity was assessed by measuring OD260/280 ratios and by examining the appearance on agarose gels. Reverse transcription and PCR amplification were performed according to manufacturers specifications using MultiScribe Reverse Transcriptase and Power SYBR Green PCR Master Mix (Applied Biosystems). Real-time PCR reactions were carried out in an ABI Prism 7500 Real-Time PCR machine (Applied Biosystems). ACT1 mRNA levels were used as controls.

Primers used to amplify CZF1 and ACT1 and PCR efficiencies were as previously described (Bassilana et al., 2005). Farnesol Production Measurements Extracellular farnesol was extracted from cell-free supernatants of stationary phase cultures grown in GPP at 30C and analyzed by gas chromatography-mass spectrometry, as described by Hornby et al (Hornby et al., 2001).

97 Results Identification of new genes required for the response to farnesol To identify important regulators of the farnesol response in C. albicans, a transposon insertion mutant library of approximately 560 mutants (Supp. Table 1) was screened for mutants defective in the morphological response to farnesol. The majority of mutants in this collection are Tn7-UAU1 plasmid insertion mutants (Nobile et al., 2006); therefore it is desirable to obtain knockout mutants for further characterization due to the possibility of partial loss-of-function mutations. Mutants that were identified as farnesol resistant in the original screen were re-streaked on individual plates to rule out interference from neighboring mutant colonies and to confirm the farnesol resistant phenotype. Eight mutants were confirmed as farnesol resistant, and they are summarized in Table 1. Mutants that were unable to interconvert between yeast and filaments (at 30 and 37, respectively) were not examined further in this study to rule out mutants that are merely defective in morphological switching and not the specific response to farnesol. No mutants were identified that were hypersensitive to farnesol, nor were any detected that demonstrated growth inhibition in the presence of farnesol.

CZF1 is required for a wild type morphological response to farnesol in both liquid and embedded conditions The czf1/czf1 mutant was identified as farnesol-resistant in the mutant library screen (Table 1) and selected for further characterization based on its known roles in morphogenesis, white/opaque cell switching, and its connection to the cAMP pathway. To test the role of Czf1 in the morphological response to farnesol, a double null

98 czf1/czf1 mutant was compared with its parental strain (CKY101), a wild type clinical isolate (SC5314), the heterozygous mutant, and the double null mutant ectopically

expressing CZF1 under the control of the MAL2 promoter in a liquid farnesol response assay (Fig. 1). In both glucose and sucrose-containing media, SC5314 and CKY101 displayed the expected reduction of early stage filaments (germ tubes) when in the presence of farnesol. However, the czf1/czf1 mutant showed only a minimal reduction of germ tubes, even in the presence of 100 M farnesol, and a haploinsufficient farnesol response phenotype was observed for the heterozygous mutant. Results were similar whether glucose or sucrose was used as a carbon source. Ectopic complementation of the czf1/czf1 mutant was able to restore the farnesol response similar to that of the heterozygous mutant in pMAL2-inducing conditions (Fig. 1B; sucrose-containing media), while there was only a minimal farnesol response in non-inducing conditions, similar to that of the czf1/czf1 mutant (Fig. 1A; glucose-containing media). These results show that the presence of Czf1 is critical to the ability of C. albicans to respond to farnesol in liquid media. Since the first described role for Czf1 was to promote filamentation in embedded conditions (Brown et al., 1999), we sought to determine whether: 1) C. albicans could respond to farnesol under such conditions and 2) Czf1 was needed for farnesol response when embedded in a matrix. For consistency with our prior work studying farnesol signaling and physiological significance (plates were incubated at 37C), we utilized defined GPP or SPP agar plates (lacking GlcNAc, see experimental procedures) in our embedded assays. Although the growth media and incubation time and temperature used in this study are different from those initially used by Brown et al (Brown et al., 1999),

99 we still observed a strong filamentation response from C. albicans SC5314 and CKY101 cells in response to growth embedded in a matrix (Fig. 2). When SC5314 and CKY101 cells were grown in embedded media containing 50 M farnesol, filamentous colonies were not observed. As with the liquid germ tube formation assays, using glucose versus sucrose as a carbon source made no difference in filamentation or farnesol response (Fig. 2). This demonstrates the capability of C. albicans to respond to farnesol in an embedded assay. As expected, the czf1/czf1 mutant exhibited a defective growth pattern in embedded conditions; filamentation was still observed, but far fewer colonies were present compared with the wild type and parental strains and colony morphology appeared different as well (Fig. 2). When the czf1/czf1 mutant was grown in agar containing farnesol, only a moderate farnesol response was observed in that the hyphae appeared shorter than in untreated samples; however, colonies were still filamentous. As with the liquid farnesol response assays, the czf1/CZF1 heterozygote maintained a haploinsufficient phenotype in both farnesol treated and untreated samples: in untreated samples, the overall level of growth was more similar to that of wild type strains, and in farnesol-treated samples, colonies were reduced in hyphal formation but still more filamentous than wild type colonies treated with farnesol. Ectopic CZF1

complementation of the czf1/czf1 mutant produced positive farnesol responses similar to that of the heterozygote in both GPP and SPP, conditions which should turn off and on, respectively, the MAL2 promoter (Fig. 2). This suggests unintended leaky promoter activation in GPP media, possibly resulting in the expression of CZF1 and complementation of the farnesol response in both GPP and SPP embedded media. These

100 results indicate that Czf1 is required for farnesol response in at least two distinct environmental conditions: liquid, aerobic growth media and embedded in a semi-solid matrix, new conditions in which to test C. albicans for a morphological farnesol response.

CZF1 mRNA levels are decreased in the presence of farnesol Since CZF1 is regulated at the mRNA level, we asked whether CZF1 mRNA levels are affected by the presence of farnesol. A time-course experiment was performed to measure CZF1 expression levels in the presence of 0, 50 or 100 M farnesol, and 40, 60, and 80 minute time points were selected based on previous studies showing the importance of this time frame to the farnesol response (Kebaara et al., 2008). As with TUP1 mRNA levels (Kebaara et al., 2008; Mosel et al., 2005), CZF1 levels were most dramatically affected 60 minutes after treatment with farnesol, showing a greater than 5fold decrease in expression relative to a sample lacking farnesol (Fig. 3B). At all 3 time points tested, both 50 and 100 M farnesol reduced CZF1 levels, relative to samples containing no farnesol (Fig. 3). These data indicate that CZF1 mRNA levels are

decreased in the presence of farnesol, during time points crucial to the farnesol response.

An efg1/ efg1 czf1/ czf1 double mutant exhibits decreased farnesol tolerance that is partially temperature dependent Upon growth of the efg1/efg1 czf1/czf1 mutant in liquid media for preparation of resting cells, an unusual temperature-regulated phenotype was observed. Mutant cells grown in YPD broth at 30C appeared small but elongated, reminiscent of

101 opaque cells (Fig. 4A), and when the efg1/efg1 czf1/czf1 double mutant was grown in YPD broth at 37C, cells appeared larger and more rounded (Fig. 4B). When these efg1/efg1 czf1/czf1 double mutant cells (pre-grown at 30C) were tested for farnesol response in the previously described mGPP/mSPP liquid germ tube formation assays (experimental procedures), we observed what appeared to be cell death. Given the importance of environmental conditions in the ability of C. albicans to survive and tolerate farnesol, methylene blue staining was performed during liquid germ tube assays to more accurately assess the level of cell death occurring in these conditions (Fig. 4C-F). For the cell death assays, resting cells were prepared as described in experimental procedures, except that cells were grown at either 30C or 37C in YPD broth until stationary phase was reached. The parental strain CAI4 was used as a control in the 30C resting cell group, and as observed for SC5314 in Langford et al (Langford et al., 2010), no significant cell death was observed in mGPP or in mSPP, even in the presence of 100 M farnesol. The czf1/czf1 and efg1/efg1 mutants (Fig. 4C-F) were similarly resistant to farnesol-mediated killing in the liquid germ tube formation assays. However, the czf1/czf1 mutant could not be tested accurately with methylene blue in these assays, due to its rapid formation of germ tubes which stain inconsistently with this dye regardless of the strain used or the presence of farnesol. The arguments for the viability of the czf1/czf1 mutant in the presence of farnesol are twofold: 1) no growth

inhibition was observed in the presence of farnesol on agar plates (data not shown) and 2) germ tube formation is a process that requires active protein synthesis (Imanishi et al., 2004) and is unlikely to occur in a dead cell. CAI4 could also not be used as a control at 37C for similar reasons; cells pre-grown at 37C start as filaments. On the other hand,

102 the efg1/efg1 czf1/czf1 double mutant and the efg1/efg1 czf1/czf1 double mutant ectopically expressing CZF1 (from the 30C resting cell group) showed extreme sensitivity to farnesol, regardless of the media used to induce CZF1 expression (Fig 4C, 4E). Conversely, when the efg1/efg1 czf1 czf1 double mutant and the efg1/efg1 czf1/czf1 double mutant expressing CZF1 under the control of the MAL2 promoter from the 37C resting cell group were tested for farnesol sensitivity, a general decrease in cell death was observed compared to the 30C resting cell group (Fig. 4C-F). Based on these results, Czf1, Efg1, and temperature appear to play important roles in farnesol tolerance, and both 30C and 37C double mutant resting cells were subsequently utilized for additional experiments in this study.

Ectopically expressing CZF1 in an efg1/ efg1

czf1/czf1

double mutant

partially restores filamentation and farnesol response in liquid and embedded conditions Can ectopic expression of CZF1 restore filamentation and response in the efg1/ efg1 czf1/czf1 double mutant? Liquid germ tube formation assays were performed on CAI4, efg1/efg1, the efg1 efg1 czf1/czf1 double mutant, and the efg1/efg1 czf1/czf1 double mutant ectopically expressing CZF1 under the control of the MAL2 promoter, and only cells that did not stain with methylene blue were counted. For the 30C resting cell group, only CAI4 was able to produce germ tubes and respond to farnesol, in both mGPP and mSPP (Fig. 5A, 5C). For the 37C resting cell group, the efg1/efg1 and the efg1/ efg1 czf1/czf1 double mutant were capable of producing very low levels of germ tubes with a slight trend of reduction in the presence of farnesol.

103 In conditions which induce CZF1 expression in the efg1/efg1 czf1/czf1 double mutant, an increase in germ tube formation was observed when compared to noninducing conditions (Fig. 5B, 5D), and farnesol reduced germ tube formation when CZF1 was expressed. When CAI4 30C resting cells were inoculated into the embedded farnesol response assays, results were similar to SC5314 and CKY101 (Fig. 6). CAI4 produced filamentous colonies in the absence of farnesol in both GPP and SPP embedded media and the presence of farnesol resulted in decreased filamentation. Note the smaller hyphae growing out from the CAI4 colonies treated with farnesol (Fig. 6); because farnesol delays filamentation rather than block it entirely, the time points selected for observation are critical. Ideally CAI4 embedded plates should have been checked at 12 h postinoculation for farnesol response (as in Fig. 2) rather than at 17h as shown (no short filaments are observed in the farnesol-treated CAI4 samples at 12 h, data not shown), but the 17 h time point was selected in order to be consistent with the other slower-growing mutant strains being tested in Fig. 6. The efg1/efg1 mutant was capable of forming filamentous colonies under embedded conditions, and a very subtle shortening of filaments was observed in farnesol-treated samples. An inoculum using 30C resting cells for the efg1/efg1 mutant is shown in Fig. 6, and similar results were obtained using an inoculum of 37C resting efg1/efg1 cells. The efg1/efg1 czf1/czf1 double mutant was capable of producing filamentous colonies in the absence of farnesol, regardless of the temperature used during resting cell preparation (Fig. 6). The

efg1/efg1 czf1/czf1 double mutant and the efg1/efg1 czf1/czf1 double mutant ectopically expressing CZF1 did not produce any colonies in the presence of farnesol

104 when 30C resting cells were used (data not shown), suggesting these cells were killed by farnesol as they were in the liquid assays. When 37C resting cells were used for the efg1/efg1 czf1/czf1 double mutant, farnesol reduced the number of colonies, but the colonies that grew were filamentous. Unexpectedly, the filamentous colonies from

farnesol-treated plates often grew in a different conformation from untreated samples. The efg1/efg1 czf1/czf1 double mutant expressing CZF1 under the control of the MAL2 promoter behaved similarly to the efg1/efg1 czf1/ czf1 double mutant in noninducing conditions (Fig. 6A), and a subtle increase in colony size and filamentation was observed in inducing media (Fig. 6B). In the presence of farnesol, these filaments were slightly shorter. Taken together, both Czf1 and Efg1 are important for farnesol tolerance in embedded conditions as well, along with temperature. When CZF1 is ectopically expressed in a strain lacking Czf1 and Efg1, the level of filamentation is partially restored in both liquid and embedded assays, and farnesol partially suppresses filamentation in both conditions.

Overproduction of farnesol is not a general quality of farnesol-resistant mutants Two of the factors known to play a role in the C. albicans morphological farnesol response, Tup1 and Nrg1, both produced 17 and 19-fold higher levels of farnesol than wild type and parental strains (Kebaara et al., 2008). In order to determine whether farnesol overproduction is a general quality of farnesol-resistant mutants, we tested the farnesol production levels of the czf1/czf1 mutant (Fig. 7). Farnesol production levels were not significantly altered in the czf1/czf1 mutant, suggesting a more specific

105 involvement of Tup1/Nrg1 in farnesol production that does not require the presence of Czf1.

106 Discussion In this study, we identified new factors involved in the C. albicans response to farnesol and have shown that Czf1 in particular plays a prominent role in the morphological response to farnesol in both liquid aerobic conditions and embedded conditions; this adds a new function for Czf1 during the quorum sensing response. Furthermore, CZF1 mRNA levels are decreased in the presence of farnesol at critical time points during the farnesol response, and unlike another factor involved in farnesol signaling (Tup1), Czf1 does not appear to regulate farnesol production levels in the cell. Surprisingly, CZF1, along with EFG1, was also shown to play an important part in farnesol tolerance; these are the first specific genes identified that control the ability of C. albicans white a/ cells to survive in the presence of farnesol. The Ras1-Cyr1-cAMP signaling pathway has been proposed to be the only direct target for farnesol in C. albicans (Davis-Hanna et al., 2008; Deveau et al., 2010). This study provides additional evidence supporting a primary role of this pathway in farnesol signaling. Here we showed that Czf1 is an important factor in the farnesol response. It has a tight regulatory relationship with Efg1, which is consistent with the Ras1-cAMP signaling pathway as a central target for farnesol. Further evidence for the significant role of cAMP signaling during the farnesol response is provided by the identification of one of the protein kinase A (PKA) isoforms in C. albicans, Tpk1, as farnesol resistant. The other PKA isoform, Tpk2, was not identified by the farnesol resistance screen as it was not present in this mutant library collection. A recent study (Deveau et al., 2010) showed that an additional phenotype of the farnesol response in C. albicans, reactive oxygen species (ROS) protection, is also mediated by the Ras1-cAMP pathway. The

107 Hog1 signaling pathway was shown to participate in ROS protection as well, but through an unknown connection between the two pathways (Deveau et al., 2010). Three

additional genes were identified that are potentially important for farnesol resistance: RLM1, YCK2, and HAP43, also function in different stress responses. These mutants can possibly be used to fill in some of the gaps between these pathways. It is intriguing that farnesol can protect the cell from ROS stress, while it can also kill opaque cells and log-phase, energy-deprived white cells. Since farnesol tolerance in C. albicans is potentially an active process (Langford et al., 2010), it is of interest to understand the key components that are involved. This study provides a new

understanding of farnesol tolerance, by identifying two factors that are required for survival in the presence of farnesol: Czf1 and Efg1. We speculate that it is no

coincidence that both of these factors also play critical roles in white/opaque cell switching (Ramirez-Zavala et al., 2008; Sonneborn et al., 1999; Srikantha et al., 2000; Vinces and Kumamoto, 2007; Zordan et al., 2007) since opaque cells are inherently more sensitive to farnesol killing than are white cells (Dumitru et al., 2007). Farnesol killed efg1/efg1 czf1/czf1 double mutant cells in liquid aerobic conditions, and it appeared to do the same in embedded media due to the lack of growth when using 30C resting cell inocula. It is unclear why single efg1/efg1 and czf1/czf1 mutants are resistant to farnesol killing, while the double mutant is overly sensitive. Czf1 and Efg1 have a synthetic effect on farnesol resistance, as farnesol tolerance was only observed in the single mutants and not the double mutant. The mere presence of CZF1 in the

efg1/efg1 czf1/czf1 double mutant (via expression of CZF1 by the MAL2 promoter) was not sufficient, however, to provide protection from farnesol induced cell death,

108 suggesting CZF1 may act upstream of EFG1 in this example. It is also possible that the presence of Czf1s native promoter is required for proper regulation in response to this particular stimulus when Efg1 is absent. Another intriguing question raised by this study is the role of temperature in farnesol tolerance. Why was the efg1/efg1 czf1/czf1 double mutant more tolerant of farnesol when grown at 37C? The answer may be tied to indirect farnesol production measurements suggesting that C. albicans produces slightly higher levels of farnesol at higher temperatures (Hornby et al., 2001). Further, there is likely some additional factor(s) that plays a minor role in farnesol tolerance and is regulated by shifts in temperature. Wor1/2 are potential candidates for such a role, in part due to their regulation of white/opaque switching (Huang et al., 2006; Zordan et al., 2006; Zordan et al., 2007). While Czf1 was shown to be part of a farnesol-resistance mechanism in C. albicans, it also strongly contributes to the morphological response to farnesol. In cells lacking Czf1 (czf1/czf1 mutants), farnesol was unable to suppress filamentation, and ectopic complementation of CZF1 in these cells restored the ability of farnesol to block germ tube formation in both liquid and embedded environmental conditions. Furthermore, in efg1/efg1 czf1/czf1 double mutants, CZF1 ectopic expression was able to partially restore filamentation and the morphological response to farnesol. These results are consistent with Czf1 functioning downstream of Efg1 during the morphological farnesol response. Through the course of testing farnesol response in conditions which either turn on or off the MAL2 promoter, we have shown that the use of glucose versus sucrose as a carbon source had no distinguishable effect on the ability of the cells to respond to farnesol.

109 The many effects of farnesol on C. albicans appear to be relatively unique to the fungus, compared with other bacterial species and fungal genera [reviewed by Langford et al (Langford et al., 2009)]. The finding that Czf1 plays a central role in the unique morphological and tolerance responses in C. albicans is consistent with the observation that Saccharomyces cerevisiae (which does not respond to farnesol) and other closely related ascomycetes lack a Czf1 homolog (Vinces et al., 2006) while other Candida species contain CZF1 homologs. As an example, the CZF1 gene in Candida

dubliniensis, a Candida species that produces the second highest farnesol levels, is farnesol tolerant, and morphologically responds to farnesol (Henriques et al., 2007; Weber et al., 2008) has the highest homology to the C. albicans CZF1 gene with 81% identity at the nucleotide level (data not shown). Furthermore, the unusually large 5 upstream region of CZF1, the CZF1 gene itself, and neighboring genes at the CZF1 locus (Vinces et al., 2006) appear to be conserved to varying degrees in additional Candida species whose sequence information has been recently made available including: Candida tropicalis, Lodderomyces guillermondii (data not shown). In conclusion, we have identified new roles for Czf1 in mediating the C. albicans tolerance to farnesol as well as farnesol-mediated filament inhibition. While the elongisporius, Candida lusitaniae, and Candida

connection of Czf1 to other factors known to play a role in the C. albicans farnesol response, such as the cAMP pathway, is apparent, others links remain unclear. For example, cross-regulation among many of the farnesol response pathways was recently summarized (Deveau et al., 2010), but little is known about the regulation of Tup1/Nrg1 and how these factors fit into the farnesol signaling network. Furthermore, other proteins

110 involved in Ras1-Cyr1-cAMP signaling such as Ras2, Cap1, and G-actin (Hall and Muhlschlegel, 2009; Zhu et al., 2009; Zou et al., 2009) have not been tested for possible roles in farnesol signaling and may yet prove to be involved. This points to the fact that farnesol can be an extremely useful tool for studying signal transduction in C. albicans by demonstrating the presence of new connections between signaling pathways.

Acknowledgements We are grateful to Aaron Mitchell, Dominique Sanglard, Carol Kumamoto, Alexander Johnson, and Gerald Fink for kindly providing the strains and plasmids used in this study. We would also like to thank Steve Harris and Gabriel Langford for use of their microscopes and cameras. This work was supported by a faculty seed grant from the Constance Miriam and Ethel Corrine Syford Memorial Fund (to A.L.A.), the HammondMaude Fling Fellowship (to M.L.L.), and the Farnesol and Candida albicans Research Fund, University of Nebraska Foundation (to K.W.N.).

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118 Weber, K., Sohr, R., Schulz, B., Fleischhacker, M., and Ruhnke, M. (2008) Secretion of E,E-farnesol and biofilm formation in eight different Candida species. Antimicrob Agents Chemother 52: 1859-1861. Westwater, C., Balish, E., and Schofield, D.A. (2005) Candida albicans-conditioned medium protects yeast cells from oxidative stress: a possible link between quorum sensing and oxidative stress resistance. Eukaryot. Cell 4: 1654-1661. Zhu, Y., Fang, H.M., Wang, Y.M., Zeng, G.S., Zheng, X.D., and Wang, Y. (2009) Ras1 and Ras2 play antagonistic roles in regulating cellular cAMP level, stationaryphase entry and stress response in Candida albicans. Mol Microbiol 74: 862-875. Zordan, R.E., Galgoczy, D.J., and Johnson, A.D. (2006) Epigenetic properties of whiteopaque switching in Candida albicans are based on a self-sustaining transcriptional feedback loop. Proc Natl Acad Sci U S A 103: 12807-12812. Zordan, R.E., Miller, M.G., Galgoczy, D.J., Tuch, B.B., and Johnson, A.D. (2007) Interlocking transcriptional feedback loops control white-opaque switching in Candida albicans. PLoS Biol 5: e256. Zou, H., Fang, H.M., Zhu, Y., and Wang, Y. (2009) Candida albicans Cyr1, Cap1 and Gactin form a sensor/effector apparatus for activating cAMP synthesis in hyphal growth. Mol Microbiol.

119 Figure Legends Figure 4-1. Czf1 is required for the morphological response to farnesol in liquid

conditions. C. albicans resting cells were inoculated into glass flasks at 106 cells/mL in either mGPP (A) or mSPP (B) broth, and 0, 50, or 100 M farnesol was added. Cultures were incubated for 1 h at 37C with shaking at 225 rpm and percentage of germ tube formation was subsequently determined (czf1/ czf1 = CKY230, czf1/CZF1= CKY116, czf1/ czf1 pMAL2-CZF1 = CKY231). Data shown are from independent experiments performed in triplicate; these were repeated with similar results on at least two separate occasions. Figure 4-2. Czf1 is required for the morphological response to farnesol in embedded media. C. albicans resting cells were mixed with either GPP (A) or SPP (B) molten agar and 0 or 50 M farnesol as described in experimental procedures, and incubated at 37C for 12 h. Independent experiments were repeated in duplicate with similar results. Figure 4-3. CZF1 mRNA expression is decreased in the presence of farnesol. SC5314

resting cells were inoculated into mGPP broth for 40, 60, or 80 minutes (A-C, respectively) with 0, 50, or 100 M farnesol and subsequently harvested for RNA extraction. Quantitative real-time PCR was used to measure relative CZF1 mRNA levels with ACT1 used as a reference gene. CZF1 levels in the minus farnesol samples were set at zero and fold change in CZF1 levels for samples with farnesol added are shown. Data are averages from three replicates. Figure 4-4. Czf1, Efg1, and temperature play a role in farnesol tolerance. The czf1/ czf1 efg1/ efg1 double mutant was grown in YPD broth at 30C (A) or 37C (B) for 24 h and differences cell morphology was observed. Scale bar = 10 m. Cells were

120 grown at either 30C or 37C in YPD broth until stationary phase was reached (24 h, except 48 h for CAI4 and efg1/ efg1 strains) to prepare resting cells as described in experimental procedures. 106 cells/mL of these resting cells were inoculated into mGPP (C,D) or mSPP (E,F) broth and 0, 50, or 100 M farnesol was added. Cultures were incubated at 37C with shaking at 225 rpm, and cell death by methylene blue staining was determined after 90 min (efg1/ efg1 = HLC67; czf1/czf1 efg1/efg1 = CKY283; czf1/czf1 efg1/efg1 pMAL2-CZF1 = AAC6). Data shown are from

independent experiments performed in triplicate; these were repeated with similar results on at least two separate occasions. Figure 4-5. Ectopic expression of CZF1 in a czf1/czf1 efg1/efg1 double mutant partially restores filamentation and farnesol response in liquid conditions. Cells were grown at either 30C or 37C until stationary phase was reached to prepare resting cells. 106 cells/mL of these resting cells were inoculated into mGPP (A,B) or mSPP (C,D) broth and 0, 50, or 100 M farnesol was added. Cultures were incubated at 37C with shaking at 225 rpm, and percentage of germ tube formation was subsequently determined. Data shown are from independent experiments performed in triplicate; these were repeated with similar results on at least two separate occasions. Figure 4-6. Czf1 and Efg1 are both required for a wild type morphological response and tolerance to farnesol in embedded agar. C. albicans resting cells (prepared at 30C or 37C) were mixed with either GPP (A) or SPP (B) molten agar and 0 or 50 M farnesol as described in experimental procedures, and incubated at 37C for 17 h. Colonies in figure were from 30C resting cells unless otherwise noted. Independent experiments were repeated in duplicate with similar results.

121 Figure 4-7. A czf1/czf1 mutant produces farnesol levels similar to that of wild type and parental strains. Cells were grown in GPP broth at 30C for 48 hours prior to farnesol extraction and quantification, as described in methods.

122 Figure 4-1. A.

Liquid Farnesol Response Assay; mGPP 100

80 % germ tube formation

60

0 FOH 50 M FOH 100 M FOH

40

20

0 SC5314 CKY101 czf1/czf1 czf1/CZF1 czf1/czf1 pMAL2-CZF1

B.
Liquid Farnesol Response Assay; mSPP 100

80 % germ tube formation

60

0 FOH 50 M FOH 100 M FOH

40

20

0 SC5314 CKY101 czf1/czf1 czf1/CZF1 czf1/czf1 pMAL2-CZF1

123 Figure 4-2

124 Figure 4-3 A.

CZF1 mRNA Levels; 40 min
Normalized Relative Fold Change in Expression 0 FOH 0 -2 -4 -6 -8 -10 -2.57 -4.08 50 M FOH 100 M FOH

B.

CZF1 mRNA Levels; 60 min

Normalized Relative Fold Change in Expression 0 FOH 0 -2 -4 -6 -8 -5.97 -10 -5.41 50 M FOH 100 M FOH

C.

CZF1 mRNA Levels; 80 min

Normalized Relative Fold Change in Expression 0 FOH 0 -2 -1.23 -4 -6 -8 -10 -1.33 50 M FOH 100 M FOH

==

125 Figure 4-4 A.

B.

C.
Cells grown at 30C; mGPP
80

60 % Cell Death

0 FOH 50 M FOH 100 M FOH

40

20

0 CAI4 efg1/efg1 czf1/czf1 czf1/czf1 efg1/efg1 efg1/efg1 pMAL2-CZF1

D.
Cells grown at 37C; mGPP
80

60 % Cell Death

40

20

0 efg1/efg1 czf1/czf1 efg1/efg1 czf1/czf1 efg1/efg1 pMAL2-CZF1

126 Figure 4-4 E.

Cells grown at 30C; mSPP
80

60 % Cell Death

40

20

0
CAI4 efg1/efg1 czf1/czf1 efg1/efg1 czf1/czf1 efg1/efg1 pMAL2-CZF1

F.
Cells grown at 37C; mSPP
80

60 % Cell Death

40

20

0
efg1/efg1 czf1/czf1 efg1/efg1 czf1/czf1 efg1/efg1 pMAL2-CZF1

127 Figure 4-5 A.

Cells grown at 30C; mGPP
100
0 FOH

B.
Cells grown at 37C; mGPP
100
50 M FOH 100 M FOH

80 60 40 20 0 CAI4

% germ tube formation

% germ tube formation

80 60 40 20 0

efg1/efg1 czf1/czf1 czf1/czf1 efg1/efg1 efg1/efg1 pMAL2-CZF1

efg1/efg1 czf1/czf1 czf1/czf1 efg1/efg1 efg1/efg1 pMAL2-CZF1

C.
Cells grown at 30C; mSPP
100 % germ tube formation 80 60 40 20 0 CAI4 efg1/efg1 czf1/czf1 czf1/czf1 efg1/efg1 efg1/efg1 pMAL2-CZF1

D.
Cells grown at 37C; mSPP
100 % germ tube formation 80 60 40 20 0 efg1/efg1 czf1/czf1 czf1/czf1 efg1/efg1 efg1/efg1 pMAL2-CZF1

128 Figure 4-6

129 Figure 4-7

Farnesol Production
2

ug FOH/g dry wt.

1.5

0.5

0
SC 53 14 CA I4

130 Table 4-1. Summary of insertion mutants with an impaired farnesol response. Mutant czf1/czf1 S. cerevisiae ortholog None Predicted or known biological processes Zinc finger transcription factor for filamentation under embedded conditions and positive regulator of white to opaque switching; binds Efg1 and expression controlled by Efg1 and Czf1 (Brown et al., 1999; Hornby et al., 2001; Ramirez-Zavala et al., 2008; Vinces et al., 2006; Vinces and Kumamoto, 2007; Zordan et al., 2007) Catalytic subunit of cAMP-dependent protein kinase A, regulator of morphogenesis; Tpk2 isoform; involved in multiple stress responses (Bockmuhl et al., 2001; Cloutier et al., 2003; Giacometti et al., 2009; Maidan et al., 2005) Transcription factor for genes involved in cell wall organization and biogenesis and various stress responses (Bruno et al., 2006; Sampaio et al., 2009) Transcription factor for amino acid permease genes (Martinez and Ljungdahl, 2005) Role in cytokinesis (Li et al., 2006) Maintenance of cell polarity, antimicrobial peptide resistance, contributes to epithelial cell damage (Park et al., 2009) Putative transcription factor Transcription factor, involved in iron limitation response (Baek et al., 2008)

tpk1/tpk1

Tpk2

rlm1/rlm1

Rlm1 Stp2 Hof1 Yck2 Hap1 Yap3

stp2/stp2 hof1/hof1 yck2/yck2

zcf14/zcf14 hap43/hap43

131 Table 4-2. Candida albicans strains and plasmids used in this study. Parental strain C. albicans strains SC5314 CAF2 CAI-4 BWP17 HLC67 (efg1/ efg1) CKY101 CKY230 (czf1/ czf1) CKY116 (czf1/CZF1) CKY231 (czf1/ czf1 pMAL2-CZF1) CKY283 (czf1/ czf1 efg1/ efg1) AAC2 Clinical isolate CAI-4 CAF2 RM1000 CAI-4 CAI-4 CAI-4 CAI-4 CAI-4 CAI-4 CKY283 Genotype/Description Source

Clinical isolate Ura+ derivative of CAI-4 ura3::imm434/ura3::imm434 ura3::imm434/ura3::imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG ura3::imm434/ura3::imm434 efg1::hisG/efg1::hisG ura3::imm434/ura3::imm434 ade2::pDB152 ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG ade2::pMAL2-URA3 ura3::imm434/ura3::imm434 CZF1/czf1::hisG -URA3-hisG ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG ade2::pMAL2-CZF1-URA3 ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG-URA3hisG efg1::hisG/efg1::hisG ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG efg1::hisG/efg1::hisG ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG efg1::hisG/efg1::hisG ade2::pMAL2-CZF1-URA3 pMAL2-CZF1 URA3 ade2 Ampr

Alexander Johnson (Gillum et al., 1984) Alexander Johnson (Leberer et al., 2001) Alexander Johnson (Fonzi and Irwin, 1993) Aaron Mitchell (Nobile and Mitchell, 2005) Gerald Fink (Lo et al., 1997) Carol Kumamoto (Brown et al., 1999) Carol Kumamoto (Vinces et al., 2006) Carol Kumamoto Carol Kumamoto (Brown et al., 1999) Carol Kumamoto (Giusani et al., 2002) This study This study

AAC6 AAC2 (czf1/ czf1 efg1/ efg1 pMAL2-CZF1) Plasmids pDB212

Carol Kumamoto (Brown et al., 1999)

132 Supplementary Table 4-1. List of mutants screened in this study. Mutant ORF# orf19.1795 orf19.7291 orf19.6760 orf19.4669 orf19.5037 orf19.11450 orf19.9364 orf19.6970 orf19.10248 orf19.9791 orf19.6952 orf19.9364 orf19.4966 orf19.7479 orf19.7412 orf19.5328 orf19.1857 orf19.8907 orf19.14178 orf19.4746 orf19.5235 orf19.3995 orf19.8635 orf19.3171 orf19.2723 orf19.768 orf19.3208 orf19.1409 orf19.564 orf19.856 orf19.11598 orf19.4284 orf19.4369 orf19.7389 orf19.4668 orf19.9508 orf19.5011 orf19.4658

133 orf19.1509 orf19.1593 orf19.695 orf19.6036 orf19.3014 orf19.3701 orf19.4763 orf19.5495 orf19.1805 orf19.13950 orf19.12265 orf19.5571 orf19.7614 orf19.10359 orf19.2990 orf19.4257 orf19.2237 orf19.771 orf19.6729 orf19.3396 orf19.2237 orf19.3563 orf19.2033 orf19.11973 orf19.3171 orf19.769 orf19.5251 orf19.7381 orf19.3202 orf19.1825 orf19.5571 orf19.580 orf19.3764 orf19.9081 orf19.5365 orf19.3012 orf19.12351 orf19.5445 orf19.6261 orf19.3009 orf19.10169

134 orf19.2763 orf19.2392 orf19.4893 orf19.2348 orf19.2653 orf19.5292 orf19.4457 orf19.6194 orf19.5094 orf19.271 orf19.1759 orf19.12603 orf19.6032 orf19.7201 orf19.9115 orf19.1005 orf19.4369 orf19.7389 orf19.4519 orf19.2660 orf19.4529 orf19.7472 orf19.6344 orf19.4428 orf19.1614 orf19.4369 orf19.3818 orf19.8837 orf19.11598 orf19.3906 orf19.7016 orf19.7400 orf19.9331 orf19.6185 orf19.3678 orf19.4535 orf19.7194 orf19.5001 orf19.4244 orf19.658 orf19.8326

135 orf19.3530 orf19.6194 orf19.6100 orf19.1510 orf19.4214 orf19.4285 orf19.3125 orf19.7208 orf19.5866 orf19.2938 orf19.9115 orf19.12706 orf19.6850 orf19.1252 orf19.5352 orf19.7451 orf19.7401 orf19.271 orf19.1394 orf19.2901 orf19.6737 orf19.811 orf19.2808 orf19.769 orf19.7207 orf19.530 orf19.4979 orf19.7381 orf19.7400 orf19.6950 orf19.14148 orf19.4409 orf19.4872 orf19.4513 orf19.6971 orf19.2270 orf19.3190 orf19.7410 orf19.4440 orf19.13704 orf19.6365

136 orf19.4116 orf19.3678 orf19.7381 orf19.3844 orf19.1331 orf19.1276 orf19.5761 orf19.7576 orf19.813 orf19.1907 orf19.4846 orf19.4543 orf19.4518 orf19.4823 orf19.7447 orf19.7324 orf19.2350 orf19.10841 orf19.5664 orf19.7610 orf19.248 orf19.425 orf19.4243 orf19.1628 orf19.3707 orf19.829 orf19.3254 orf19.1291 orf19.6365 orf19.4969 orf19.2061 orf19.1793 orf19.3122 orf19.5887 orf19.4188 orf19.4139 orf19.1795 orf19.5100 orf19.6267 orf19.7583 orf19.11659

137 orf19.7451 orf19.4474 orf19.3012 orf19.6036 orf19.4743 orf19.6763 orf19.4010 orf19.7207 orf19.2508 orf19.5016 orf19.5023 orf19.998 orf19.7355 orf19.814 orf19.411 19.6817 19.3809 19.7247 19.11410 19.4433 19.5908 19.7150 19.12215 19.12786 19.4972 19.173 19.4125 19.4766 19.3187 19.3986 19.6680 19.5026 19.4670 19.4318 19.7359 19.723 19.7518 19.2745 19.7583 19.5729 19.7570

138 19.3127 19.4568 19.1135 19.1187 19.5548 19.3308 19.2647 19.6182 19.431 19.7318 19.5380 19.1497 19.1255 19.3305 19.4573 19.9191 19.1007 19.2280 19.5338 19.4767 19.4776 19.4778 19.13396 19.2356 19.9326 19.909 19.2054 19.2260 19.798 19.889 19.2331 19.2315 19.1178 19.2393 19.2399 19.2458 19.3088 19.2612 19.2674 19.1358 19.1496

139 19.3193 19.3300 19.1589 19.1565 19.3405 19.3407 19.1694 19.1729 19.1826 19.3683 19.3753 19.3835 19.6781 19.6845 19.6850 19.7025 19.10266 19.10478 19.1227 19.1822 19.2064 19.2077 19.2432 19.2623 19.3390 19.4649 19.487 19.567 19.6985 19.718 19.735 19.976 orf19.3966 orf19.3966 orf19.299 orf19.299 orf19.4887 orf19.4887 orf19.2623 orf19.2623 orf19.1563

140 orf19.1563 orf19.1563 orf19.3010.1 orf19.3010.1 orf19.3010.1 orf19.4884 orf19.4884 orf19.3434 orf19.3434 orf19.2613 orf19.2613 orf19.2613 orf19.5412 orf19.4981 orf19.4981 orf19.4981 orf19.1277 orf19.1277 orf19.5867 orf19.5867 orf19.3869 orf19.3869 orf19.3869 orf19.1490 orf19.1490 orf19.1490 orf19.2476 orf19.2476 orf19.2476 orf19.5674 orf19.893 orf19.532 orf19.532 orf19.1714 orf19.3642 orf19.7251 orf19.3193 orf19.7374 orf19.166 orf19.2647 orf19.3188

141 orf19.1358 orf19.1499 orf19.6817 orf19.681 orf19.4662 orf19.5848 orf19.6407 orf19.5975 orf19.1032 orf19.2356 orf19.6985 orf19.7371 orf19.4766 orf19.3012 orf19.5133 orf19.5917 orf19.6203 orf19.861 orf19.6038 orf19.5849 orf19.4649 orf19.3294 orf19.2623 orf19.2315 orf19.1069 orf19.3190 orf19.6102 orf19.723 orf19.2540 orf19.3434 orf19.4961 orf19.5953 orf19.6124 orf19.173 orf19.6121 orf19.217 orf19.1035 orf19.1718 orf19.2423 orf19.255 orf19.2748

142 orf19.2753 orf19.2808 orf19.3252 orf19.3405 orf19.3876 orf19.4145 orf19.4166 orf19.4225 orf19.4288 orf19.4450 orf19.4524 orf19.5924 orf19.5940 orf19.7372 orf19.6227 orf19.6713 orf19.7319 orf19.5995 orf19.5759 orf19.4979 orf19.2395 orf19.6626 orf19.7523 orf19.1813 orf19.2217 orf19.702 orf19.7388 orf19.7388 orf19.896 orf19.896 orf19.7001 orf19.7001 orf19.4866 orf19.4866 orf19.5181 orf19.5181 orf19.4252 orf19.4252 orf19.4084 orf19.4084 orf19.469

143 orf19.469 orf19.5350 orf19.5350 orf19.2605 orf19.2605 orf19.5224 orf19.5224 orf19.663 orf19.663 orf19.451 orf19.451 orf19.1874 orf19.1874 orf19.4242 orf19.4242 orf19.2395 orf19.2395 orf19.3047 orf19.3047 orf19.794 orf19.794 orf19.4892 orf19.4892 orf19.5408 orf19.5408 orf19.223 orf19.223 orf19.3256 orf19.3256 orf19.1196 orf19.1196 orf19.835 orf19.835 orf19.469 orf19.469 orf19.4084 orf19.4084 orf19.2341 orf19.2341 orf19.35 orf19.35

144 orf19.4001 orf19.4001 orf19.4867 orf19.4867 orf19.7281 orf19.7281 orf19.3744 orf19.6369 orf19.4144 orf19.4269 orf19.4909 orf19.4909 orf19.4890 orf19.4890 orf19.4308 orf19.4308 orf19.4432 orf19.4432 orf19.3545 orf19.3545 orf19.7510 orf19.7510 orf19.2678 orf19.6913 orf19.6913 orf19.5068 orf19.5068 orf19.3854 orf19.3854 orf19.5357 orf19.4347 orf19.4347 orf19.7510 orf19.7510 orf19.5224 orf19.5224 orf19.3751 orf19.3049 orf19.7044 orf19.7044 orf19.5253

145 orf19.5253 orf19.2277 orf19.2277 orf19.7451 orf19.7451 orf19.3530 orf19.3530 orf19.4297 orf19.4297 orf19.4518 orf19.4518 orf19.2268 orf19.2102 orf19.3256 orf19.3256 orf19.844 orf19.844 orf19.3841 orf19.3841 orf19.3049 orf19.3049 orf19.2436 orf19.846 orf19.846 orf19.7355 orf19.3720 orf19.3720 orf19.2910 orf19.2910 orf19.4002 orf19.428 orf19.428 orf19.1341 orf19.3415 orf19.3415 orf19.3415 orf19.6243 orf19.7523 orf19.7523 orf19.6889 orf19.6889

146 orf19.5901 orf19.5901 orf19.1341 orf19.1341 orf19.130 orf19.130 orf19.2222 orf19.1283 orf19.1283 orf19.1283 orf19.1283 orf19.7510 orf19.7510 orf19.5911 orf19.5911 orf19.7164 orf19.7164 orf19.5325 orf19.5325 orf19.895 orf19.895 orf19.5162 orf19.5162 orf19.7652 orf19.7652 orf19.460 orf19.460

147

CHAPTER 5 Summary and Future Directions With a brief excerpt from: Langford, M.L., Atkin, A.L., and Nickerson, K.W. (2009) Cellular Interactions of Farnesol, a Quorum Sensing Molecule Produced by Candida albicans. Future Microbiol. 4: 1353-62.

148 Summary Farnesol has effects on all eukaryotic cells tested, and for many cell types, a low level of farnesol is sufficient to inhibit growth or induce cell death. In contrast, C. albicans has evolved mechanisms to produce and tolerate higher levels of farnesol and use it as a quorum sensing molecule. A current challenge is to develop a unifying model for the mode of action for farnesol in eukaryotes and elucidate the mechanisms used by C. albicans to evade the adverse effects of farnesol and respond to farnesol as a signaling molecule. In this study, we established environmental conditions in which C. albicans is able to resist farnesol-mediated cell death: stationary phase cells provided with an energy source. Conversely, we have defined conditions that stimulate sensitivity to farnesol in C. albicans: energy-deprived, log phase cells (Chapter 2). Using the aforementioned environmental conditions that allow C. albicans to tolerate farnesol and use it as a signaling molecule, two novel factors were identified that are involved in the morphological response to farnesol: Tup1 and Czf1 (Chapters 3, 4). It seems that farnesol suppression of filamentation during C. albicans quorum sensing works through both positive and negative regulators of filamentation (Czf1 and Tup1, respectively) to achieve the resulting change in morphology. Czf1 also played a dual role in the farnesol response, as it was partially responsible for farnesol tolerance in quorum sensing conditions.

Farnesol Tolerance and Sensitivity in C. albicans The precise mechanism for C. albicans white cell farnesol tolerance during quorum sensing remains unclear. Since the effects of farnesol on other organisms have

149 been studied in seventeen different species (summarized in Chapter 1), this is an important mechanism to understand in the field of interspecies communication. Furthermore, as exogenous farnesol has many effects in the mammalian host during infection such as preventing infection of mucosal surfaces, increasing virulence in a disseminated infection and interference of the host cytokine response [Chapter 1 and (Navarathna et al., 2007)], understanding the farnesol tolerance mechanism in C. albicans may prove useful therapeutically. It is unknown how C. albicans white cells are resistant to the toxic effects of farnesol. Do the cells have a type of passive resistance, such as changes in membrane composition, or do they undergo active resistance, such as conversion of farnesol into a non-toxic compound? In order to look for genes that may be involved in farnesol tolerance, a screening approach was used (Chapter 4). However, the screen was

unsuccessful in identifying mutants that were growth-inhibited (sensitive) in the presence of farnesol, even at levels as low as 10 M. Even so, the mutant library collection used in this study is by no means a complete representation of the entire collection of C. albicans predicted open reading frames (ORFs); there are 6197 predicted ORFs in the genome, compared with the 560 mutants contained within the mutant library collection used, leaving plenty of room for future identification of farnesol tolerance factors as more mutants become available. The identification of Czf1 and Efg1 as important players in farnesol survival is intriguing because a farnesol-sensitive phenotype was observed only when both genes were deleted. Ectopic expression of CZF1 in the efg1/efg1 czf1/czf1 double mutant could not provide complete protection from farnesol killing yet the single efg1/efg1

150 and czf1/czf1 mutants were unharmed (Chapter 4). Perhaps Czf1 functions upstream of Efg1 in this situation, or the normal regulation of CZF1 might be especially important in the absence of EFG1. In the reverse situation, could ectopic expression of EFG1 in the double mutant restore partial or full farnesol resistance? Czf1 and Efg1 likely regulate a redundant gene or set of genes for farnesol tolerance because the single mutants do not show a farnesol sensitive phenotype while the double mutant exhibits a synthetic farnesol sensitivity phenotype. While changes in gene expression have been extensively studied in the efg1/efg1 mutant compared to wild type/parental strains (Braun and Johnson, 2000; Harcus et al., 2004; Sohn et al., 2003), there is no matching data for Czf1 available. Microarray analyses designed to complement existing data on Efg1 may reveal genes that are commonly regulated by Czf1 and Efg1, either directly or indirectly. Genes whose expression was altered in cells lacking Czf1 and Efg1 could also be compared to existing microarray data comparing gene expression between white and opaque cells (Lan et al., 2002; Tsong et al., 2003; Zhao et al., 2005). Genes that are differentially expressed in opaque cells, efg1/efg1, and czf1/czf1 mutants would comprise a list of strong candidates for potential farnesol resistance factors. One further step that may be taken would be to look at other farnesol-sensitive organisms to see if homologs to the candidate farnesol resistance genes are present in those species. The role of temperature in farnesol tolerance (the efg1/efg1 czf1/czf1 double mutant was more sensitive at 30C than at 37C) may be correlated to evidence suggestive of slightly elevated farnesol production levels at higher temperatures (Hornby et al., 2001). It would be informative to examine changes in gene expression and to measure farnesol production in the efg1/efg1 czf1/czf1 double mutant at both 30C

151 and 37C, compared to wild type and parental strains. Differentially regulated genes may be partly responsible for the cellular morphology and farnesol resistance changes observed at these two temperatures. One would also predict that farnesol production may be significantly lowered at 30C based on the degree of farnesol sensitivity for the double mutant cells grown at this temperature. Analysis of the efg1/efg1 single mutant would need to be included in this study; initial results using this mutant resulted in high variation between replicates (M.L. Langford, data not shown) and further replicates will need to be conducted to determine whether Efg1 plays a role in farnesol production. Furthermore, a possible role for Tup1 in farnesol resistance should not be ignored. As tup1/tup1 mutants produced 17-fold more farnesol compared to wild type and parental strains, these cells may have heightened abilities to tolerate farnesol. However, it will be difficult to study higher tolerance to farnesol in these mutant cells since they are locked in the filamentous morphology (unless secondary mutations are introduced), unlike the efg1/efg1 and czf1/czf1 mutants. Preliminary experiments must first be conducted to better understand the tup1/tup1 farnesol resistance, such as a comparison of farnesol tolerance between yeast, pseudohyphal, and hyphal cells. If the loss of Tup1 results in higher farnesol tolerance, would a triple efg1/czf1/tup1 mutant have restored resistance? What would the farnesol production levels in that mutant be? It is intriguing that all three of these factors play a prominent role in white-opaque switching, farnesol production and/or farnesol tolerance, as well as the morphological response to farnesol.

152 Signaling During the Morphological Response to Farnesol in C. albicans Because the presence of a single farnesol receptor has still not been identified, it remains a possibility that there may be multiple targets for farnesol in the cell. Two models (network and central processor) have been proposed with regard to C. albicans signaling in general (Braun and Johnson, 2000) and are worth considering when examining the farnesol response. That study suggested C. albicans follows the network signaling model, whereby many individual connections are formed between the different regulatory pathways in the cell. This conclusion was reached when they observed that several hyphal-specific genes are not regulated in unison in different hyphal inducing conditions but in fact respond as individuals. If network signaling is indeed occurring during the farnesol response, farnesol could help identify many of the different branches in these pathways. The Ras1-cAMP pathway has been identified as a critical, even a possible direct target for farnesol inhibition during yeast to filament inhibition by farnesol (Davis-Hanna et al., 2008; Deveau et al., 2010). Although a recent study (Davis-Hanna et al., 2008) showed the inability of the ras1/ras1 mutant to respond to farnesol in aerobic conditions, it remains undetermined if it is capable of responding to farnesol under different environmental conditions, specifically during embedded growth. The

ras1/ras1 mutant is defective in filamentation under aerobic conditions and embedded in YPS (yeast extract, peptone, sucrose) agar (Maidan et al., 2005), but it has not been tested in our GPP embedded media. If filamentation can be achieved in our embedded assay, the morphological response to farnesol can be tested; this would both complement

153 the existing aerobic data and determine the importance of Ras1 to the farnesol response in other environmental conditions. A recently characterized protein, Ras2 (Zhu et al., 2009), plays an antagonistic role with Ras1 regarding stationary phase entry, stress resistance, and cellular cAMP levels. Although Ras2 shares poor sequence homology with Ras1 and phylogenetic analyses have suggested it belongs to a divergent group of fungal Ras proteins (Ras2 has a homolog with high sequence similarity to C. dubliniensis), it still has functional GTPase activity (Zhu et al., 2009). More importantly, since Ras2 has an antagonistic effect on many of the physiological roles of Ras1, it may somehow be involved in farnesol signaling as well. The ras2/ras2 mutant maintains the ability to make

filaments in aerobic and embedded conditions, making it an ideal candidate for testing the morphological response to farnesol. Might Ras2 play an antagonistic response to Ras1 during farnesol signaling as well? While one new factor involved in the morphological response to farnesol, Czf1, has a known connection to the Ras1-cAMP pathway in C. albicans, it remains unknown how another factor involved, Tup1, fits into this model (Chapters 3, 4). Further studies using farnesol as a tool may decipher whether Tup1 is somehow connected to the cAMP pathway or is independently involved in the farnesol response. Construction of a triple efg1/czf1/tup1 mutant may prove useful, not only to study farnesol tolerance as mentioned earlier, but to study farnesol signaling as well. If this mutant is viable, what would its morphology be in different growth conditions? Would this mutant be capable of responding to farnesol? Vectors that can be used to delete EFG1, CZF1, and TUP1 have already been constructed, as well as ectopic expression constructs for all three genes

154 (Bockmuhl and Ernst, 2001; Braun and Johnson, 1997; Brown et al., 1999; Lo et al., 1997), a couple of which have already been obtained by the Atkin laboratory. Therefore, all the necessary tools are available to create not only a triple mutant, but the remaining double mutant combinations (i.e. tup1/czf1, efg1/tup1) and ectopically express each gene in the desired mutant strains. Construction and analysis of these strains can provide valuable insight into C. albicans morphological signaling networks. For example, if TUP1 was ectopically expressed in the efg1/efg1 czf1/czf1 double mutant background and no differences in farnesol response were observed, Tup1 would most likely be acting upstream of these two factors. Other ways to test for a possible connection between Tup1 and the Ras1-cAMP pathway would be to determine whether Czf1 and/or Efg1 are responsible for the increase in TUP1 mRNA levels and/or Tup1 protein levels observed during the morphological response to farnesol. For example, are the TUP1 mRNA levels in a czf1/czf1 mutant background still increased during farnesol treatment? If the mRNA levels do not increase in response to farnesol, it would suggest that Czf1 is either directly or indirectly responsible for the regulation of Tup1 during the farnesol response, and this information could potentially provide the first data regarding the regulation of Tup1 since regulatory factors upstream of Tup1 remain unknown. Regardless of whether Czf1 would specifically regulate TUP1 expression, followup experiments designed to better understand which genes are controlled by Czf1 are desirable for the C. albicans research field. While it has been shown to bind its own promoter and negatively regulate its expression, it is unknown what other genes are directly regulated by Czf1. Moreover, because it controls so many processes in C.

155 albicans: morphogenesis, white-opaque switching, farnesol tolerance, and the

morphological response to farnesol, it is of considerable interest to identify genes that are directly regulated by this unique transcription factor, both without and in the presence of farnesol. ChIP (chromatin immunoprecipitation)-Seq, would be an ideal procedure to determine direct targets of Czf1. Prior to this experiment, however, Czf1 antibodies would ideally be raised (all protein work concerning Czf1 has utilized an HA-tagged Czf1 protein, which would have the potential to slightly alter the natural function of the protein). The ChIP-Seq method would first cross-link DNA (chromatin)-protein

complexes and shear the DNA into smaller pieces. An antibody against Czf1 can be used to immunoprecipitate DNA bound with Czf1, and deep sequencing could subsequently be used to sequence the precipitated DNA. Sequence analyses would finally provide the location of the Czf1 binding sites in the C. albicans genome. In addition to CZF1, the mutant screen from Chapter 4 identified 7 other genes that may play a role in the farnesol response. Of note, identification of STP2 was a bit of a surprise, as it is the only gene identified with no direct ties to morphogenesis or stress responses. Either the acquisition of amino acids plays a more important role during farnesol signaling than is currently appreciated, or Stp2 may participate in farnesol signaling in an undiscovered manner. The RLM1 and YCK2 genes are both attractive candidates for further analysis, particularly when examining the farnesol survival mechanism, due to their known roles in stress resistance.

156 Final Thoughts One common theme that can be discerned from studying the farnesol response in C. albicans is that farnesol affects a variety of regulatory factors within the polymorphic fungus. This unique feature has allowed us to study the interrelatedness of some of the signaling networks in an effort to better understand the genetic pathways present in the opportunistic pathogen. Farnesol can be used as an instrument to dissect the complicated and intertwined pathways present in C. albicans, including pathways yet to be fully understood, such as the regulation of Tup1, Hog1 signaling, and stress response signaling. Although farnesol was initially discovered as a quorum sensing molecule that only regulates morphogenesis, the discovery of its many other functions will hopefully allow for a more complete understanding of C. albicans signaling as a whole.

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